WO2009139599A2 - Protéine recombinante p27 à cellule perméable, un polynucléotide codant cette protéine et une composition anticancéreuse la contenant en tant que principe actif - Google Patents

Protéine recombinante p27 à cellule perméable, un polynucléotide codant cette protéine et une composition anticancéreuse la contenant en tant que principe actif Download PDF

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WO2009139599A2
WO2009139599A2 PCT/KR2009/002588 KR2009002588W WO2009139599A2 WO 2009139599 A2 WO2009139599 A2 WO 2009139599A2 KR 2009002588 W KR2009002588 W KR 2009002588W WO 2009139599 A2 WO2009139599 A2 WO 2009139599A2
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recombinant protein
cell
mtd
seq
amino acid
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PCT/KR2009/002588
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Korean (ko)
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WO2009139599A3 (fr
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조대웅
임정희
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주식회사 프로셀제약
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Publication of WO2009139599A3 publication Critical patent/WO2009139599A3/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention relates to a p27 recombinant protein in which a macromolecular transduction domain (MTD) is fused to a tumor suppressor p27, a polynucleotide encoding the cell permeable p27 recombinant protein, the cell
  • MTD macromolecular transduction domain
  • Cancer occurs through genetic and epigenetic changes from normal cells. These cancer cells have a survival factor such as IGF (insulin-like growth factor) and do not cause apoptosis, resulting in abnormal cellular intestinal and division; Activation of vascular endothelial growth factor (VEGF) inducers promotes angiogenesis; Cell metastasis is induced by the reduction of E-cadherin, which is involved in cell-to-cell junctions.
  • cancer cells may induce excessive cell proliferation and growth due to mutations in cell cycle regulatory genes or malfunction of regulatory proteins during their cell division, thereby promoting tumorigenesis.
  • the cell cycle is a series of processes in which one cell divides and divides into two cells in sequence. Many factors are involved in cell cycle regulation. One of the most important is cyclin-dependent kinase (CDK). Cyclin-dependent Kinases regulate each stage of the cell cycle by binding to cyclins in each cell cycle to form functional units and phosphorylating them. This process results in the formation of a cyclin-CDK complex that is activated at each stage of the cell cycle. The formation of cyclin-CDK complexes is regulated by a variety of mechanisms, particularly CDK inhibitors that inhibit CDK activation (Balomenos, D and Martinez, AC, Immunol. Today 21: 551-5, 2000)
  • CDK inhibitors inhibit unwanted cell proliferation in vivo through the activity of CDK, which is a family of INK4 (inhibitors of CDK4), a specific inhibitor capable of recognizing only complexes of CDK4 and 6 and cyclin D, and cyclin E-CDK2, cyclin It is divided into two groups of CIP / KIP families that inhibit the activity of A / B-CDK2.
  • the INK4 family includes pl6, pl5, pl8, pl9, and the like, and the CIP / KIP family includes p21, p27, and p57.
  • P27 known as the dual cyclin-dependent kinase inhibitor 1B or CDKN1B, is a cell cycle inhibitory protein that binds to CDK and cyclin, respectively, and regulates cell division progression at G1.
  • DNA sequences are similar and have structural similarities to them at the protein level, which have functional properties that bind to different classes of cyclin and CDK molecules.
  • p27 has been reported to inactivate the cell cycle by inhibiting complex formation such as cyclin E / CDK2 and cyclin A / CDK2 (Drexler HC, Cell Cycle 2 (5): 438-41, 2004).
  • p27 binds to cyclin D-CDK4 / 6 in the early G1 phase of the cell cycle and promotes its entry into the nucleus to increase the stability of cyclin D, while cyclin in Gl-arrested cells where the cell cycle is stopped Strong binding to the E-CDK2 complex Inhibition inhibits catalytic activity.
  • such negative regulation inhibits cell proliferation by inhibiting the progression from the G1 phase to the S phase, thereby inhibiting tumor formation.
  • p27 functions as a tumor suppressor. This is confirmed by studies showing increased cell proliferation and pituitary tumor formation in mice with the p27 gene removed.
  • breast carcinoma, gastric cancer, colon cancer, prostate adenocarcinoma, non-small cell lung carcinoma, cervical adenocarcinoma, glioblastoma It has been reported that p27 expression is reduced in various human cancers such as glioblastoma (Kau et al., Nat. Rev. Cancer 4: 106-1 17, 2004; Belletti B et al., Cwr. Med. Chem) 12 (14): 1589-605, 2005).
  • the present inventors are convinced that if they can induce the overexpression of p27 in the body or deliver the p27 protein to the body directly with high efficiency, tumor formation can be effectively suppressed.
  • Macromolecules are fused with "hydrophobic macromolecule transduction domain (MTD)" and other intracellular carriers, synthesized, expressed, purified in recombinant protein form, and delivered into cells. It can be accurately and quickly transported to a specific location, effectively fulfilling the many roles required: Likewise, large molecule delivery domains (MTDs) are fused to peptides, proteins, DNA, RNA, synthetic compounds, and cannot enter the cell. It enables the delivery of many impermeable materials.
  • MTD macromolecule transduction domain
  • the present inventors prepared a p27 recombinant protein fused with a macromolecule transfer domain to a tumor suppressor p27 to impart cell permeability, and the recombinant protein p27 was isolated from outside the cell in vitro as well as in vitro.
  • the present invention has been completed by confirming that it can be effectively used as an anticancer agent to treat p27 defect or loss of function occurring in various cancers of humans by effectively delivering into the nucleus of cells. [Detailed Description of the Invention]
  • an object of the present invention is to provide a cell-permeable p27 recombinant protein as an anticancer agent that can treat p27 defect or loss of function caused by various cancers of humans by imparting cell permeability to tumor suppressor p27 and introducing it into cells with high efficiency.
  • the present invention provides a cell-permeable p27 recombinant protein which is imparted cell permeability by the fusion of tumor suppressor p27 and macromolecular transduction domain (MTD) to introduce p27 into the cell with high efficiency. do.
  • the present invention also provides a polynucleotide encoding the cell permeable p27 recombinant protein.
  • the present invention provides an expression vector comprising the polynucleotide and the transforming bacteria transformed with the expression vector.
  • the present invention also provides a method for producing a cell permeable p27 recombinant protein comprising culturing the transforming bacteria.
  • the present invention provides a pharmaceutical composition for anticancer drug for treating cell permeable p27 defect or loss of function containing the p27 recombinant protein as an active ingredient.
  • the p27 recombinant protein having cell permeability introduces a tumor suppressor p27 into the nucleus of the cell with high efficiency, thereby preventing the formation of a complex of cyclin-dependent kinase and cyclin and inhibiting the cell cycle of cancer cells, thereby preventing unnecessary cell proliferation in vivo. It inhibits, thereby inducing the death of cancer cells can be usefully used as an anticancer agent for a variety of human cancers.
  • Figure 3 is a result of PCR amplification of the p27 recombinant protein fused JO-58 and JO-68 MTDS to the full-length p27 of the full-length form in accordance with the present invention
  • Figure 4a is a pGEM-T Easy vector of FIG. Subcloning PCR Amplification Products
  • FIG. 4b shows that the PCR amplification product of FIG. 3 was subcloned into the pGEM-T Easy vector
  • Figure 5a is a schematic diagram showing the process of producing a recombinant expression vector by cloning the p27 recombinant fragment of the present invention in a pET-28a (+) vector,
  • FIGS. 7A and 7B show the result of purifying p27 recombinant protein in which JO-58 MTD and JO-68 MTD were fused to p27 in full-length form expressed from the recombinant transformed vector of the present invention
  • 8a and 8b are the results of analyzing the cell permeability of p27 recombinant protein fused with JO-58 MTD and JO-68 MTD in full-length p27 according to the present invention, respectively,
  • 9A and 9B show the results of observing the cell permeability of p27 recombinant protein fused with JO-58 MTD and JO-68 MTD to full-length p27 according to the present invention in confocal laser scanning microscope in mouse fibroblasts,
  • Figure 10a is the result of analyzing the intracellular function of the cell permeable p27 recombinant protein according to the present invention by Western blotting in HCT-1 16 cell line, a human colon cancer cell line
  • 10B is a result of Western blotting analysis of the intracellular function of the cell-permeable p27 recombinant protein of the present invention in the MDA-MB-231 cell line, which is a human breast cancer cell line.
  • Figure 12a is a result of observing the viability of cancer cells after treatment of the cell-permeable p27 recombinant protein fused with JO-58 MTD and JO-68 MTD to full-length p27 in human breast cancer cell line MCF7 for 3 days,
  • 12b is a result of observing the viability of cancer cells after treatment for one day and cell cultured p27 recombinant protein fused with JO-58 MTD to full-length p27 in human breast cancer cell line MCF7 for 1 day,
  • FIG. 13 shows the results of apoptosis induction of the cell permeable p27 recombinant proteins HM lP 27 and 111 0) 271 according to the present invention
  • FIG. 14 shows the cell permeable p27 recombinant proteins HM lP 27 according to the present invention.
  • HM lP 27M which was investigated through intracellular Annexin-V staining
  • Figure 15a is a result of measuring the change in tumor size every day after subcutaneous injection of the cell-permeable p27 recombinant proteins HM lP 27 and ⁇ 27 ⁇ , according to the present invention into the tumor-forming mice for 9 days,
  • Figure 15b is a photograph comparing the tumor size extracted from the mouse 14 days after the administration of the cell-permeable p27 recombinant proteins HM lP 27 and HM 1 p27M 1 according to the present invention by subcutaneous injection for 9 days
  • Figure 16a is a result of measuring the change in tumor size daily after intravenous injection of the cell-permeable p27 recombinant proteins HM lP 27 and ⁇ 27 ⁇ , according to the present invention into the tumor-forming mice for 14 days,
  • Figure 16b is a photograph comparing the tumor size extracted from the mouse 7 days after the administration of the cell permeable p27 recombinant protein HM lP 27 and ⁇ ! ⁇ ?” by intravenous injection for 14 days,
  • FIG. 17 shows the effect of apoptosis-inducing apoptosis in tumor tissues of mice injected with subcutaneous injections of the cell permeable ⁇ 27 recombinant proteins HM lP 27 and HM lP 27Mi according to the present invention by TUNEL (TUNEL) analysis. Observed.
  • the present invention provides a cell-permeable p27 recombinant protein (CP-p27) and a poly encoding the same, which are endowed with cell permeability through the fusion of a tumor suppressor p27 with a macromolecular delivery domain (MTD).
  • CP-p27 cell-permeable p27 recombinant protein
  • MTD macromolecular delivery domain
  • a feature of the present invention is the macromolecular delivery domain (hereinafter referred to as "MTD") specific for the tumor suppressor p27, which is a macromolecule that is not readily introduced into cells using macromolecular intracellular delivery technology ( ⁇ ). Abbreviated) to impart cell permeability to deliver p27 into cells with high efficiency.
  • the macromolecular delivery domain can then be fused only to one end of p27, or to both ends thereof.
  • Intracellular delivery technology using MTD a hydrophobic peptide derived from secreted protein, enables real-time quantitative regulation of p27 concentration in vivo, mediating the delivery of p7 to cancer tissue and It can be distributed into cancer cells.
  • This effect is attributed to the p27-induced cyclin-dependent kinase in cancer tissues.
  • By inhibiting the complex formation of cyclin and inhibiting the cell cycle of cancer cells it is possible to suppress unnecessary cell proliferation in vivo, thereby providing an environment that can induce the death of cancer cells.
  • a p27 recombinant protein was developed by fusing the p27 to a peptide domain that enables the delivery of macromolecules into cells as a macromolecular delivery domain (MTD) that can be fused to a tumor suppressor p27.
  • MTD macromolecular delivery domain
  • cell permeable p27 recombinant protein includes a macromolecular delivery domain and a tumor suppressor p27, and means a covalent complex formed by genetic fusion or chemical bonding thereof.
  • genetic fusion is meant a linear covalent linkage formed through the genetic expression of a DNA sequence encoding a protein.
  • Tumor suppressor P27 which inhibits unnecessary cell proliferation in vivo by inhibiting the cell cycle of cancer cells, is a tumor suppressor having a nucleotide sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2, a cell cycle that regulates cell division progression in G1 phase It acts as an inhibitory protein.
  • Tumor suppressor P 27 consists of a domain comprising amino acids 1 to 198 in the amino acid sequence of SEQ ID 2 (see FIG. 2).
  • a polypeptide having cell permeability including an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 3 to 196 may be used.
  • Macromolecular delivery domains comprising any of the amino acid sequences set forth in SEQ ID NOs: 3-196 can mediate the influx of biologically active molecules comprising polypeptides, protein domains, or full-length proteins into cells through cell membranes. Cell permeable polypeptide.
  • Macromolecular delivery domains according to the present invention are characterized in that N-terminal region, hydrophobic region and It is designed to have a hydrophobic region that forms helix from signal peptides consisting of three parts of the secreted protein cleavage site at the end, thereby imparting cell membrane targeting activity.
  • These macromolecular delivery domains can directly penetrate the cell membrane without damaging the cell, allowing the target protein to move into the cell to achieve its desired function.
  • Macromolecular delivery domains having an amino acid sequence of SEQ ID NOs: 3 to 196 that can be fused to tumor suppressor p27 according to the present invention are shown in Tables la to li below.
  • the cell permeable p27 recombinant protein of the present invention is a macromolecular delivery domain, in which one of the 194 MTDs is fused to one or both ends of the tumor suppressor p27, and to one end of the fusion construct.
  • SV40 large T antigen-derived nucleus batch sequence It may have a structure in which a histidine-tag (His-Tag) affinity domain is fused for localization sequence (NLS) and easy purification.
  • ABPP amyloid beta A4 protein precursor
  • 'MTD 2 ' secreted protein derived from Streptomyces coe / co / or
  • JO- is a macromolecular delivery domain.
  • Three permeable length forms are devised for each of these as a cell permeable p27 recombinant protein using 58 MTD and JO-68 MTD.
  • full length form refers to a form comprising an intact amino acid sequence that does not include the deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 2 of tumor suppressor p27. it means.
  • p27 derivatives containing various modifications by deletion, addition, insertion or substitution of one or more amino acid residues in its amino acid sequence within the range not impairing the anticancer activity of p27 can be used in the present invention. have.
  • Hp27M 2 His-p27-MTD 2 fused JO-68MTD at its C-terminus
  • SEQ ID NO: 198 has an amino acid sequence of which SEQ ID NO: is encoded by a polynucleotide having a nucleotide sequence of 197;
  • His-p27-MTD Has an amino acid sequence of SEQ ID NO: 200, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 199;
  • His-MTDi-p27-MTD! ( ⁇ 27 ⁇ ,) has the amino acid sequence of SEQ ID NO: 202, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 201.
  • His-MTD 2 -p27 has an amino acid sequence of SEQ ID NO: 204, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 203;
  • His-p27-MTD 2 (Hp27M 2 ) has the amino acid sequence of SEQ ID NO: 206, which is encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 205;
  • His-MTD 2 -p27-MTD 2 (HM 2 p27M 2 ) has an amino acid sequence of SEQ ID NO: 208, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 207.
  • Hp27 His-p27
  • This control protein is represented by SEQ ID NO: 210. Having an amino acid sequence, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 209.
  • the present invention also provides a recombinant expression vector comprising a polynucleotide encoding the cell permeable p27 recombinant protein and a transformed bacterium transformed with the recombinant expression vector.
  • a "recombinant expression vector” refers to a gene construct that is capable of expressing a target protein or target RNA in a suitable host cell, and includes a gene regulatory element operably linked to allow the gene insert to be expressed.
  • operably linked refers to a function in which the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein or RNA of interest are functionally linked to perform a general function.
  • a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the encoding nucleic acid sequence.
  • Operational linkage with recombinant expression vectors can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.
  • Expression vectors usable in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include membrane targeting or in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers. It may be prepared in various ways according to the purpose, including a signal sequence or leader sequence for secretion. The promoter of the expression vector may be constitutive or inducible.
  • the expression vector is a host cell containing the vector It contains a selection marker for selection, and the origin of replication if the expression vector is replicable.
  • the recombinant expression vector of the present invention prepared as described above may be, for example, pET28a (+)-HM, p27.
  • the recombinant expression vector pET28a (+)-HM lp 27 is a cell permeable p27 recombinant according to the present invention at the Ndel restriction enzyme site in the multi cloning site (MCS) of pET-28a (+) vector (Novagen, Germany)
  • MCS multi cloning site
  • pET-28a (+) vector Novagen, Germany
  • nucleotide of the present invention is cloned into a pET-28a (+) vector (Novagen, USA) having a His-Tag sequence.
  • the cell-permeable p27 recombinant protein expressed from the recombinant expression vector is fused with one of JO-58 MTD and JO-68 MTD at one or both ends of p27 in full-length form, and histidine-labeled and NLS at its N-terminus. It is connected.
  • the present invention also provides a transformed bacterium transformed with the recombinant expression vector.
  • the transforming bacterium of the present invention may preferably be Escherichia coli, and E. coli is a recombinant expression vector of the present invention, for example, a cell-permeable recombinant protein in which JO-58 MTD is fused to the p27 N-terminus of the full length of the present invention.
  • pET28a (+)-HM LP 27 containing nucleotides encoding HM, p27, a large amount of cell permeable p27 recombinant protein can be expressed.
  • Transformation includes any method of introducing a nucleic acid into a host cell, and may be performed by transformation techniques known to those skilled in the art.
  • transformation techniques known to those skilled in the art.
  • microprojectile bombardment electric shock Electroporation, calcium phosphate (CaP0 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, PEG-mediated fusion, microinjection and liposome-mediated methods ), but is not limited to such.
  • HM lP 27 fused JO-58 MTD to the p27 N-terminal of the full length prepared by the above method and HM lP 27M fused JO-58 MTD to the p27 sock end of the full length Transgenic bacteria obtained by transfecting the recombinant expression vector containing E. coli DH5a with each of them were dated May 6, 2009 at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology. Deposited with KCTC-11 5 0 7 BP and KCTC-11508BP.
  • the present invention also provides a method for producing a cell permeable p27 recombinant protein comprising culturing the transgenic bacteria.
  • the production method is performed by culturing the transformed bacteria under appropriate medium and conditions so that the polynucleotide encoding the cell permeable p27 recombinant protein of the present invention is expressed in the recombinant expression vector introduced into the transforming bacterium of the present invention.
  • the method for culturing the transgenic bacteria to express the recombinant protein is known in the art, for example, inoculated into a suitable medium in which the transgenic bacteria can grow, and then cultured, and then inoculated into the culture medium. And expression of the protein by culturing in the presence of suitable conditions such as isopropyl- ⁇ -D-thiogalactoside (IPTG), a gene expression inducing agent.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • substantially pure recombinant protein can be recovered from the culture.
  • substantially pure refers to a recombinant protein of the present invention and a sequence of polynucleotides encoding the same. Means substantially no other protein derived from the host cell.
  • Recovery of the recombinant protein expressed in the transgenic bacterium can be carried out through various separation and purification methods known in the art, and the cell lysate is usually removed to remove cell debris, culture impurities, and the like. After centrifugation, precipitation, for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (precipitation of protein fractions using acetone, ethane, etc.) can be performed, and dialysis, electrophoresis And various column chromatography and the like.
  • recombinant proteins expressed in bacteria transformed with the recombinant expression vector may be classified into soluble fraction and insoluble fraction according to the characteristics of the protein during protein separation. If most of the expressed protein is in the soluble fraction, the protein can be isolated and purified without difficulty according to the method described above. However, if most of the expressed protein is in the form of insoluble fraction, that is, inclusion body, it is centrifuged after dissolving the maximum protein with a solution containing protein denaturant such as urea and surfactant. It can be purified by performing dialysis, electrophoresis and various column chromatography column.
  • the structure of the protein by the solution containing the protein denaturant Desalting and refolding steps are required during the purification of proteins from insoluble fractions because they can be modified and lose their activity. That is, the desalting and reconstituting may be performed by dialysis and dilution using a solution containing no protein denaturant or centrifugation using a filter. In addition, even when the protein is purified from the soluble fraction, when the salt concentration in the solution used for purification is high, these desalting and regeneration steps may be performed.
  • the insoluble fraction such as Triton X-100
  • Triton X-100 is used. It is dissolved in a buffer containing a nonionic surfactant, treated with ultrasound and centrifuged to obtain a precipitate. The obtained precipitate is dissolved in a solution containing denaturing agent urea and centrifuged to obtain a supernatant.
  • the recombinant protein of the present invention most dissolved from the insoluble fraction using urea is purified using a histidine-binding purification kit, and the purified protein is then removed from the salt by dialysis using a dialysis membrane or the like and protein structure.
  • the recombinant protein of the present invention can be obtained by performing the nativeization process of.
  • the present invention provides a pharmaceutical composition for anticancer drugs for treating p27 defect or loss of function containing the cell-permeable p27 recombinant protein as an active ingredient.
  • the cell-permeable p27 recombinant protein according to the present invention efficiently introduces the tumor suppressor p27 into the nucleus of the cell, thereby preventing the formation of the complex of cyclin-dependent kinase and cyclin. By inhibiting the cell cycle of cancer cells, unnecessary cell proliferation in vivo can be suppressed.
  • the p27 recombinant protein with cell permeability is useful as an anticancer agent for preventing and / or treating a variety of human cancers by inducing apoptosis of cancer cells through reactivation of signaling mechanisms related to cell cycle inhibition and apoptosis induction. Can be used.
  • composition containing the recombinant protein according to the present invention as an active ingredient may further comprise a pharmaceutically acceptable carrier, such as a carrier for oral administration or a carrier for parenteral administration.
  • a pharmaceutically acceptable carrier such as a carrier for oral administration or a carrier for parenteral administration.
  • Carriers for oral administration include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like.
  • the recombinant protein according to the present invention can be used in the form of ingested tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups and wafers in combination with excipients.
  • carriers for non-oral administration include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutane.
  • Other pharmaceutically acceptable carriers may be used by reference to those described in the following references (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
  • compositions according to the invention can be formulated in a variety of parenteral or oral dosage forms.
  • Representative of non-oral dosage forms are injectable formulations, preferably aqueous isotonic solutions or suspensions.
  • injectable formulations may be prepared according to techniques known in the art, using suitable dispersing or wetting agents and suspending agents.
  • each component may be formulated for injection by dissolving in saline or serous solution.
  • oral dosage forms include, for example, tablets and tablets, and these formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, textose, sucrose, manny, solbi, cellulose and / or glycine).
  • diluents e.g., lactose, textose, sucrose, manny, solbi, cellulose and / or glycine
  • the tablet may include binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, Disintegrants, absorbents, colorants, flavors and / or sweeteners, such as alginic acid or its sodium salt.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, Disintegrants, absorbents, colorants, flavors and / or sweeteners, such as alginic acid or its sodium salt.
  • the formulations may be prepared by conventional mixing, granulating or coating methods.
  • compositions of the present invention may further comprise auxiliaries such as preservatives, hydrating agents, emulsifiers, salts for controlling osmotic pressure and / or laxatives and other therapeutically useful substances, and may be formulated according to conventional methods.
  • auxiliaries such as preservatives, hydrating agents, emulsifiers, salts for controlling osmotic pressure and / or laxatives and other therapeutically useful substances, and may be formulated according to conventional methods.
  • the route of administration of the composition according to the present invention can be administered to humans and animals orally or parenterally, such as intravenous, subcutaneous, intranasal or intraperitoneal. Oral administration also includes sublingual application. Parenteral administration includes injections and drip methods such as subcutaneous injection, intramuscular injection, intravenous injection, direct tumor injection.
  • the total effective amount of the recombinant protein of the present invention may be administered to a patient in a single dose, and the fractionated treatment protocol in which multiple doses are administered for a long time. It may also be administered by.
  • the composition of the present invention may vary the content of the active ingredient depending on the extent of the disease, but can be repeatedly administered several times a day at an effective dose of 5 to 20 rag when administered once per adult.
  • the effective dose of the recombinant protein may be determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the drug. .
  • composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is shown.
  • Recombinant proteins of EGFP-MTE ⁇ and EGFP-MTD 2 purified in soluble form were fluorescently labeled with FITC (fluorescein-5-isothiocyanate, Molecular Probe).
  • FITC 1 ⁇ at a concentration of 333 m / concentration was mixed with 2 to 20 m of recombinant protein, and then bound with shaking for 2 hours at room temperature, avoiding light.
  • recombinant protein of EGFP-MTD 2 was dialyzed in DMEM medium at 4 ° C. for 1 day to remove unlabeled FITC, and the recombinant protein recovered therefrom was Bradford.
  • the concentration was analyzed by (Bradford) protein quantitation. As a result, the concentration of each recombinant protein was measured to be about 1 fig / ⁇ .
  • RAW 264.7 cells derived from macrophages from mice were injected with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (penicillin / streptomycin, WelGENE) 500 mg. / MEM was inoculated in DMEM medium (WelGENE) and incubated under wet conditions of 37 ° C, 5% C0 2 .
  • FBS fetal bovine serum
  • penicillin / streptomycin penicillin / streptomycin
  • each of the FITC-labeled EGFP-MTE and EGFP-MTD 2 recombinant proteins prepared above was treated with RAW 264.7 cells at a concentration of 10 ⁇ and further incubated for 1 hour at 37 ° C. Subsequently, trypsin / EDTA (T / E, Invitrogen) was treated to remove free FITC exposed to the cell membranes of the RAW 264.7 cells treated with the recombinant protein, and washed three times with cold PBS (phosphate buffered saline). It was. Prepared cells were analyzed by FACScan flow cytometry (Becton Dickinson, Calif.) Using CellQuest Pro cytometric analysis software.
  • the cell permeability of the EGFP-MTDi and EGFP-MTD 2 recombinant proteins according to the present invention is expected to be non-cell permeable (scrambled MTD), positive control (kFGF4-derived MTD), FITC- already confirmed cell permeability Cell permeability was determined using the fluorescence single treatment group and the protein group not treated with the comparison group.
  • the recombinant protein fused with JO-58 MTD and JO-68 MTD as a macromolecular delivery domain according to the present invention showed a higher level of plasma membrane permeability than the control group. It was.
  • the gray curved surface is the cell alone, and the black curve is the FITC.
  • the blue curve alone shows a non-cell permeable negative control (scrambled MTD)
  • the red curve shows a cell permeable positive control (kFGF4-derived MTD)
  • the green curve shows the recombinant proteins of JO-58 MTD and JO-68 MTD, respectively.
  • NIH3T3 cells derived from mouse fibroblasts to visually identify intracellular delivery sites of JO-58 MTD and JO-68 MTD whose cell permeability was primarily confirmed by flow cytometry in Example ⁇ 1-1>.
  • FITC-labeled EGFP-MTE ⁇ and EGFP-MTD 2 recombinant proteins were treated at a concentration of 10 ⁇ M and cultured at 37 ° C for 1 hour in a Korean-American Cell Line Bank, Seoul, Korea. (confocal laser scanning microscopy) was observed.
  • the NIH3T3 cells used were incubated for 24 hours in 8-well chamber slides (LabTek, Nalgen Nunc).
  • NIH3T3 cells were cultured in DMEM medium containing 10% FBS and 5% penicillin / streptomycin 500 m / mC.
  • the cultured cells were washed three times with PBS, then at 37 ° C, 5% C0 2 with serum free DMEM, serum free DMEM containing FITC, or serum free DMEM containing 10 ⁇ each of FITC-labeled recombinant protein. It was treated for hours. After 1 hour, the cells were fixed by treating with 4% paraformaldehyde for 20 minutes at room temperature.
  • the immobilized cells were confirmed to be delivered to the nucleus and cell permeable through the PI (propidium iodide, Sigma-Aldrich) counterstaining staining to fluoresce the nucleus so as to easily distinguish the MTD intracellular delivery site. After staining with ⁇ for 5 minutes at 1 concentration, the cells were washed three times with PBS. Slide mounting medium with 10 ⁇ DABCO (Fluca) containing polyvinyl alcohol to preserve fluorescent label of intracellular recombinant protein After 15 minutes of instillation, observation was performed with confocal laser scanning microscope.
  • PI sodium iodide
  • the confocal laser scanning microscope observed the prototype, FITC fluorescence and PI fluorescence of the cells using a Normaski filter, and the FITC was excited at 488 nm and detected with a 530 ran bandpass filter. It was.
  • the recombinant protein fused JO-58 MTD and JO-68 MTD as a macromolecular delivery domain according to the present invention is a cell alone, FITC alone, negative control (scrambled MTD) and positive control (kFGF4-derived MTD) It was confirmed that it is widely distributed in the nucleus compared to the control. Intracellular nuclear localization of the JO-58 MTD and JO-68 MTD recombinant proteins is proportional to the relative cell permeability results of the flow cytometry.
  • Example ⁇ 1-2> In order to confirm whether the cell permeability of the JO-58 MTD and JO-68 MTD of the present invention, which were confirmed in cell permeability, was cultured in Example ⁇ 1-2>, the following experiment was performed It was.
  • FIG. 2 Three full-length forms were devised for each MTD as follows to prepare cell-permeable p27 recombinant protein using JO-58 MTD (MTD and JO-68 MTD (MTD 2 )) (FIG. 2):
  • Hp27M 2 His-p27-MTD 2 fused JO-68 MTD at its C-terminus
  • PCR polymerase chain reaction
  • the forward and reverse primers for amplification of ⁇ 27 ⁇ have the nucleotide sequences of SEQ ID NOs: 213 and 214, respectively;
  • Forward and reverse primers for amplification of ⁇ 2 ⁇ 27 have the nucleotide sequences of SEQ ID NOs: 215 and 212, respectively;
  • Forward and reverse primers for amplification of ⁇ 27 ⁇ 2 have the nucleotide sequences of SEQ ID NOs: 211 and 216, respectively;
  • the forward and reverse primers for the amplification of 13 ⁇ 4 ⁇ 2 27! ⁇ 2 have the nucleotide sequences of SEQ ID NOs: 215 and 216, respectively.
  • PCR reaction was a template gene 100 ng of human p27 cDNA, dNTP complex (dGTP, dATP, dTTP and dCTP, 2 mM each), 0.5 ⁇ each primer, lOx Taq complete solution 10 ⁇ , Taq polymerization
  • a solution containing 0.5 fiH of enzyme (Takara, Japan) was carried out with a final volume of 100 fd semi-agar.
  • PCR reaction conditions were first denatured for 2 minutes at 95 ° C, followed by 30 cycles of 45 seconds at 95 ° C, 45 seconds at 67 ° C and 45 seconds at 72 ° C, and finally at 72 ° C. Amplified for 5 minutes at. 0.8% agarose after reaction Electrophoresis was performed on the gel to confirm the amplified product. As shown in Figure 3, it was confirmed that each recombinant fragment fused MTD was amplified to the desired size.
  • the pGEM-T Easy vector into which each p27 recombinant protein gene was inserted was digested at 37 ° C for 2 hours using restriction enzyme Ndel to obtain respective recombinant fragments. After recovering the recombinant fragments in the agarose gel, each of them was extracted and purified using a commercially available kit (QIAquick Gel extraction kit; Qiagen, US A). Meanwhile, the expression vector pET-28a (+) vector (Novagen, USA) with histidine-tag and T7 promoter was cleaved under the same conditions as above using restriction enzyme Ndel.
  • FIG. 5A shows the recombinant fragment inserted into the pET-28a (+) vector with Ndel restriction enzyme. By electrophoresis on 0.8% agarose gel, from which it was confirmed that each recombinant fragment was correctly inserted into the vector.
  • Recombinant protein expression vector thus obtained was pET28a (+)-HM lP 27, pET28a (+) -Hp27M l5 pET28a (+)-HM, p27Mi, pET28a (+)-HM 2 p27, pET28a (+)-Hp27M 2 And pET28a (+)-HM 2 p27M 2 , among which the recombinant expression vector pET28a (+)-HM lP 27 and pET 2 8a (+)-HM 2 p27M 2 were obtained for transformation of E. coli DH5a.
  • the bacteria DH5a / HM lP 27 and ⁇ (! / ⁇ ?) were deposited on May 6, 2009 at the Biological Resource Center in the Korea Research Institute of Bioscience and Biotechnology. Deposited with 11507BP and KCTC-11508BP.
  • the full-length p27 recombinant protein prepared by using JO-58 MTD, His-MTE ⁇ -pS? has the amino acid sequence of SEQ ID NO: 198, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 197; His-p27-MTD, (HP27M,) has an amino acid sequence of SEQ ID NO: 200, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 199; 1 ⁇ - ⁇ 110 ⁇ 27-1 ⁇ 10, (HM lP 27M0 has an amino acid sequence of SEQ ID NO: 202, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 201.
  • a full-length ⁇ 27 recombinant protein prepared using JO-68 MTD, His-MTD 2 -p27 has an amino acid sequence of SEQ ID NO: 204, which is the base sequence of SEQ ID NO: 203 Encoded by a polynucleotide having a; His-p27-MTD 2 (Hp27M 2 ) has the amino acid sequence of SEQ ID NO: 206, which is determined by a polynucleotide having the nucleotide sequence of SEQ ID NO: 205 Coded; His-MTD 2 -p 2
  • the MTD 2 (HM 2 p27M 2 ) has an amino acid sequence of SEQ ID NO: 208, which is encoded by a polynucleotide having a base sequence of SEQ ID NO: 207.
  • control of the cell permeable recombinant protein p27 Heath T Dean the full-length p27 was prepared His-p27 (Hp27) that are labeled only the fusion.
  • This control protein has an amino acid sequence of SEQ ID NO: 210, which is encoded by a polynucleotide having a base sequence of SEQ ID NO: 209.
  • Example 3 Expression of Recombinant Protein
  • each of the recombinant expression vectors prepared in Example ⁇ 2-1> was thermally stratified to each of the E. coli strains BL21 (DE3), BL21 Gold (DE3), BL21 CodonPIus (DE3), and BL21 Gold (DE3) pLysS.
  • the cells were cultured in LB medium containing 50 kanamycin.
  • E. coli introduced with the recombinant protein gene was then inoculated in 1 LB medium and incubated overnight at 37 ° C, which was then inoculated in 100 LB medium and cultured at 37 ° C until OD 600 reached 0.6-0.7. .
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • Escherichia coli BL21 identified as the optimal strain in Example ⁇ 3-1>
  • Recombinant Expression Vector in CodonPlus (DE3) pET28a (+) -Hp27M b pET28a (+)-HMip27M !, pET28a (+) -HM 2 p27, pET28a (+)-Hp27M 2 and pET28a (+)-HM 2 p27M 2, respectively
  • the cells were cultured in LB medium containing 50 ug / i of kanamycin.
  • E. coli into which the recombinant protein gene was introduced was inoculated in 25 LB medium and incubated overnight at 37 ° C, it was inoculated again in 1f LB medium until the OD 600 reached 0.6 to 0.7 at 37 ° C.
  • the cell-permeable p27 recombinant protein of the present invention having a size of about 25 kDa is in inclusion body in the form of most insoluble fractions. It was confirmed that the expression of the target protein was significantly increased in the IPTG-treated medium (+) compared to the IPTG-free medium ( ⁇ ).
  • BL21 CodonPlus (DE3) strains transformed with each of the recombinant expression vectors of the present invention were cultured in 1 £ LB medium as in Example 3.
  • the cells obtained by centrifugation of each culture were suspended in 20 lye complete layers (100 mM NaH 2 P0 4 , 10 mM Tris-HCl, 8 M urea, pH 8.0), being careful not to bubble.
  • Cells were crushed at low temperature using an ultrasonic homogenizer equipped with a microtip.
  • the processing time set the output of the device to 25 o / o of the maximum output, and the treatment was repeated for 10 seconds after 45 minutes treatment for 7 minutes.
  • the lysed inclusion body was centrifuged at 4,000 xg for 20 minutes at 4 ° C to remove the precipitate and the supernatant was recovered.
  • the recovered supernatant was loaded onto Ni-NTA agarose resin imparted with Nitrilotriacetic acid agarose. At this time, Ni-NTA agarose was used after washing and equilibrating several times with a complete dissolved solution.
  • the supernatant was adsorbed onto the resin with gentle stirring with a shaker at 4 ° C for at least 8 hours.
  • reaction solution containing the recombinant protein was centrifuged for 5 minutes at 1,000 rpm per minute at 4 ° C for 5 minutes to remove the reaction solution, and the resin was washed to remove non-specific adsorbents. 5 washes with mM NaH 2 P0 4 , 10 mM Tris-HCl, 8 M urea, pH 6.3).
  • Elution buffer Elution buffer: 100 mM NaH 2 P0 4 , 10 mM Tris-HCl, 8 M urea, pH 4.5) was loaded and the protein was eluted by stirring at least 2 hours, or 8 hours, on a shaker.
  • Electrophoresis was performed on a 12% SDS-PAGE gel to assay the purity of the eluted protein, and then the gel was gently shaken with Coomassie brilliant blue R and the band of the target protein was clear. Discoloration was carried out using the bleach solution.
  • the recombinant protein of the present invention purified from the insoluble fraction as in Example 4 was denatured by 8 M urea, which is a potent denaturant, the restitution process was performed as follows to convert it to the active form.
  • the purified recombinant protein was refolded buffer (0.55 M guanidine HC1, 0.88 M L-arginine, 50 mM tris-HC1, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 1 mM oxidization).
  • Recombinant protein was reactivated, i.e., reconstituted by dialysis at 4 ° C for at least 72 hours using glutathione oxidized and 1 mM reduced glutathione reduced). At this time, every 24 hours, the superimposition solution in the container was exchanged.
  • the activated recombinant protein was then dialyzed with a dialysis membrane (Snakeskin pleated, PIERCE) for 9 hours at 4 ° C. against Dulbecco's Modified Eagle Medium (EM) with 1% penicillin / straptomycin added to the cell culture medium. It was. At this time, every 3 hours in the container DMEM was exchanged. The cell-permeable p27 recombinant protein converted to the active form by the reprogramming process was used in subsequent experiments.
  • PIERCE Dulbecco's Modified Eagle Medium
  • FACS fluorescence-activated cell sorting
  • the cell-permeable p27 recombinant protein isolated and purified in soluble form was fluorescently labeled using FITC (fluorescein-5-isothiocyanate, Molecular Probe). 2 to 20 mg of the recombinant protein was mixed with FITC 1 ⁇ at a concentration of 333 gM, and then bound while shaking for 2 hours at room temperature to avoid light. Fluorescently labeled cell permeable p27 recombinant protein was dialyzed in DMEM medium at 4 ° C. for 1 day to remove unlabeled FITC, and the recombinant protein recovered therefrom was analyzed by Bradford protein assay. It was. As a result, the concentration of each recombinant protein was measured to be about 1.
  • FITC fluorescein-5-isothiocyanate
  • RAW 264.7 cells derived from macrophages from mice were treated with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (WelGENE) 500 mg. / m £ containing EM medium (WelGENE) and incubated under 37 ° C, 5% C0 2 wet conditions.
  • FBS fetal bovine serum
  • WelGENE penicillin / streptomycin
  • each of the prepared FITC-labeled cell permeable p27 recombinant proteins (HM, p27, Hp27M l5 HMip27M h HM 2 p27, Hp27M 2 and HM 2 p27M 2 ) was treated in RAW 264.7 cells at a concentration of 10 ⁇ 37 1 hour more at ° C Incubated.
  • trypsin / EDTA T / E, Invitrogen
  • Example ⁇ 6-1> ⁇ 3 ⁇ 3 cells derived from mouse fibroblasts (Korea Cell Line Bank, Seoul, South Korea) treated with FITC-labeled cell permeable p27 recombinant protein (HM lP 27, Hp27M l5 HMip27M b HM 2 p27, Hp27M 2 and HM 2 p27M 2 ) at a concentration of 10 ⁇ and at 37 ° C. After incubation for 1 hour, observation was performed with confocal laser scanning microscopy.
  • FITC-labeled cell permeable p27 recombinant protein HM lP 27, Hp27M l5 HMip27M b HM 2 p27, Hp27M 2 and HM 2 p27M 2
  • NIH3T3 cells used were incubated for 24 hours in 8-well chamber slides (LabTek, Nalgen Nunc). At this time, NIH3T3 Cells were cultured in DMEM medium containing 500 mg / ⁇ of 10% FBS and 5% penicillin / straptomycin. The cultured cells were washed three times with PBS, then at 37 ° C, 5% C0 2 with serum free DMEM, serum free DMEM containing FITC, or serum free DMEM containing 10 ⁇ each of FITC-labeled recombinant protein. It was treated for hours. After 1 hour, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature.
  • the immobilized cells were confirmed to be delivered to the nucleus and cell permeable through a PI (propidium iodide, Sigma-Aldrich) counterstain that stains the nuclear nucleus to facilitate differentiation of MTD intracellular delivery sites. After staining with PI for 5 min at 1 / g / m £ concentration, cells were washed three times with PBS. In order to preserve the fluorescent label of the intracellular recombinant protein, 10 ⁇ DABCO (Fluca) -containing polyvinyl alcohol-mounting medium was added onto the slide, followed by confocal laser scanning microscope after 15 minutes.
  • PI sodium iodide
  • the confocal laser scanning microscope observed the prototype, FITC fluorescence and PI fluorescence of the cells using a Normaski filter, and the FITC was excited at 488 nm and detected by a 530 nm bandpass filter. Became.
  • the FITC-labeled cell permeable p27 recombinant protein according to the present invention is more widely distributed in the nucleus than cells alone, FITC alone and MTD-free control.
  • Intracellular nuclear localization of the cell-permeable p27 recombinant protein fused to JO-58 MTD or JO-68 MTD is proportional to the relative cell permeability results of the flow cytometry, which results in cells of the cell-permeable p27 recombinant protein of the present invention. Permeability is once again demonstrated.
  • the cell-permeable p27 recombinant protein according to the present invention and a non-MTD-fused p27 protein (Hp27) as a control group were each treated at 500 ⁇ g in 20 ⁇ M concentrations, respectively, and then 37 ° in which 5% CO 2 was supplied for 2 hours. Recoil in C incubator. After 2 hours of treatment, cells were washed twice with PBS and incubated for 9 hours under the same conditions in the presence of serum.
  • cells were crushed for 30 minutes on ice using 100 ⁇ lysis buffer (20 mM HEPES, pH 7.2, 1% Triton-X, 10% glycerol and proteinase inhibitor).
  • 100 ⁇ lysis buffer (20 mM HEPES, pH 7.2, 1% Triton-X, 10% glycerol and proteinase inhibitor).
  • the cell lysates were collected by heating the obtained cell lysate at 100 ° C for 10 minutes and then adding the recombinant protein to the SDS-PAGE loading buffer at a concentration of 20 ⁇ . It was prepared and stored at -20 ° C until use.
  • phospho-MEK1 / 2 phospho-MEKl / 2, Ser217 / 221, 45 kDa, Cell Signaling Technology
  • phospho-Erk Thr202 / Tyr204, 42/44 kDa, Cell Signaling Technology
  • cleaved caspase-3 cleaved caspase-3, Asp 175, 17 kDa, 19 kDa, Cell Signaling Technology
  • Anti-mouse IgG-HRP goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology
  • anti-rabbit IgG-HRP anti-rabbit IgG-HRP, Santa Cruz Biotechnology
  • the cell lysates quantified above were subjected to electrophoresis at 100 V on an SDS-PAGE gel for 1.5 hours, and then transferred to PVDF membrane at 100 V for 1.5 hours. Transcription membranes were 5% (w / v) dissolved in TBS / T complete solution (10 mM Tris-CI, pH 8.0, 150 mM NaCl, 0.05% Twen 20) to prevent nonspecific adsorption with antibodies. After blocking for 1 hour in powdered milk, each of the primary antibodies was added and reacted at 4 ° C. for 1 day.
  • the membrane was then washed 5 times with TBS / T buffer and then secondary antibody was added to react for 1 hour at room temperature, and repeated washing 5 times with TBS / T complete layer, and ECL (enhanced chemiluminescence, GE) for detecting chemiluminescence.
  • ECL enhanced chemiluminescence, GE
  • Healthcare Amersham UK reagent was used to detect and analyze antigens.
  • p27 increased the expression of p21 causing cell cycle arrest and the expression of cleaved caspase-3, a marker of apoptosis, while increasing the cell cycle of the tumor.
  • Activating MEK phosphorylation (P-MEK) and Erk phosphorylation (P-Erk) decreased.
  • HM lP 27 and ⁇ 27 ⁇ ! It was confirmed that the recombinant protein exhibited superior anticancer activity by inhibiting the cell cycle of the cultured cancer cell line and reducing tumor formation.
  • Human colon cancer cell line HCT-116 (Korea Cell Line Bank) was run on RPMI 1640 medium (L-glutamine 300 mg / €, 25 mM HEPES, 25 mM NaHC0 3 89.3%, heat-inactivated fetal bovine serum 9.8%, streptomycin / penicillin 0.9%). Incubated in a 37 ° C incubator supplied with 5% CO 2 . 2 RPMI 1640 medium per well was added to the 6-well plate and the cells cultured above were inoculated therein and then incubated at 37 ° C. for 1 day.
  • Each well was treated with the cell permeable p27 recombinant protein of the present invention (11 ⁇ 27, ⁇ 27 ⁇ , ⁇ 27 ⁇ , ⁇ 2 ⁇ 27, ⁇ 27 ⁇ 2 , and ⁇ 2 ⁇ 27 ⁇ 2 ) at 10 ⁇ concentrations, and then RPMI 1640 with serum present. Exchange with medium and incubate at 37 ° C for 16 h. After the incubation was terminated, the cancer cells were fixed with formaldehyde and stained with crystal violet.
  • cell permeable p27 recombinant protein of the present invention in particular JO-58 MTD bound HM lP 27, Hp27M ! And HM, p27M, significantly reduced the proliferation of cancer cells compared to the control protein (Hp27). From this, it was confirmed in vitro that the proliferative ability of cancer cells can be effectively inhibited by the cell permeable p27 recombinant protein of the present invention.
  • the human breast cancer cell line MCF7 (Korea Cell Line Bank) RPMI
  • 1640 medium L-glutamine 300 mg / e, 25 mM HEPES, 25 mM NaHC0 3 89.3%, Inactivated fetal calf serum 9.8%, straptomycin / penicillin (0.9%) was incubated in a 37 ° C incubator supplied with 5% CO 2 . 2 1 RPMI 1640 medium per well was added to a 6-well plate and inoculated therein with the cells cultured above, followed by incubation at 37 ° C. for 1 day.
  • HM lP 27, Hp27M ls ⁇ 27 ⁇ !, HM 2 p27, Hp27M 2 , and HM 2 p 2 7M 2 a cell permeable p27 recombinant protein (HM lP 27, Hp27M ls ⁇ 27 ⁇ !, HM 2 p27, Hp27M 2 , and HM 2 p 2 7M 2 ) each day for 3 days at 10 ⁇ concentrations. After the incubation was terminated, the cancer cells were fixed with formaldehyde and stained with crystal vial.
  • the cell-permeable p27 recombinant protein of the present invention in particular, JO-58 MTD-bound HM lP 27, ⁇ 27 ⁇ , and HM 1 P27M] was treated compared to the control protein ( ⁇ 27).
  • the proliferation of cancer cells was found to be markedly reduced.
  • the cell permeability is JO-58 MTD coupled to each well of a 6-well plate, the cells are cultured as described above ⁇ 27 recombinant protein HM lP 27, ⁇ 2 VI! And processes the HM lP 27Mi respectively 10 ⁇ concentration and 37 ° After 1 day incubation in C, the cells were washed twice with PBS and incubated for 2 days under the same conditions in the presence of serum. After the incubation was terminated, the cancer cells were fixed with formaldehyde and stained with crystal vial.
  • Human colon cancer cell line HCT-1 16 (Korea Cell Line Bank) was prepared using RPMI 1640 medium (L-glutamine 300 mg / ⁇ , 25 mM HEPES and 25 mM NaHC0 3 89.3%, heat inactivated fetal bovine serum 9.8%, streptomycin / penicillin 0.9 %) was used to incubate in a 37 ° C incubator supplied with 5% C0 2 .
  • RPMI 1640 medium containing 2 ⁇ FBS per well and inoculated at a concentration of 5xl0 6 cells / of each of the cells HCT-1 16 cultured above.
  • the well plate was incubated at 37 ° C. for 1 day to allow cells to grow while adhering to the well plate. After removing the medium, the cells attached to the well plate were washed with cold PBS.
  • JO-58 MTD-coupled cell permeable p27 recombinant protein (HM lP 27, Hp27M, and ⁇ 27 ⁇ ,) and MT27-unfused p27 protein (Hp27) were treated in each well at 20 ⁇ concentration according to the present invention. It was then reacted in a 371 incubator fed with 5% CO 2 for 2 hours. After 2 hours treatment, cells were washed twice with PBS and incubated for 9 hours under the same conditions in the presence of serum.
  • the cells were quenched for 30 minutes on ice using 100 ⁇ lysed permeate (20 mM HEPES, pH 7.2, 1% Triton-X, 10% glycerol and proteinase inhibitor). Collected by pulverization, the obtained cell lysate was heated for 10 minutes at 100 ° C and then the recombinant protein was added to the SDS-PAGE loading buffer at a concentration of 20 ⁇ cell lysate samples was prepared and stored at -20 ° C until use.
  • 100 ⁇ lysed permeate 20 mM HEPES, pH 7.2, 1% Triton-X, 10% glycerol and proteinase inhibitor.
  • caspase-7 35 kDa, Cell Signaling Technology
  • cleaved caspase-7 Asp 198, 20 kDa, Cell Signaling Technology
  • primary antibodies anti-mouse IgG- Goat anti-mouse IgG-HRP, Santa Cruz Biotechnology
  • anti-rabbit IgG-HRP goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology
  • cell lysates quantified above were subjected to electrophoresis for 1.5 hours at 100 V on an SDS-PAGE gel, and then transcribed at 100 V for 1.5 hours with PVDF membrane.
  • Transcription membranes were 5% (w / v) dissolved in TBS / T complete solution (10 mM Tris-CI, pH 8.0, 150 mM NaCl, 0.05% Twen 20) to prevent nonspecific adsorption with antibodies. After blocking for 1 hour in powdered milk, each of the primary antibodies was added and reacted at 4 ° C. for 1 day. After that, the membrane was washed 5 times with TBS / T complete solution, and then the reaction was repeated at room temperature for 1 hour with the addition of a secondary antibody, and repeated 5 times with TBS / T buffer, and the ECL (enhanced chemiluminescence, GE) for chemiluminescence detection was repeated. Healthcare Amersham UK) reagent was used to detect and analyze antigens.
  • TBS / T complete solution 10 mM Tris-CI, pH 8.0, 150 mM NaCl, 0.05% Twen 20
  • caspase-7 a marker of apoptosis in these cultured cancer cells
  • Each well was treated with ⁇ 27 and ⁇ 2 VI fused with JO-58 MTD as a recombinant protein, and ⁇ 27 without MTD fused at 20 ⁇ M, respectively, and then cultured in serum-free medium for 2 hours. After incubation, the cells were washed twice with cold PBS and the cells were suspended in lx binding buffer at 1 ⁇ 10 6 cells / concentration. The cell suspension 100 was transferred to an EP tube, and 5 m of Annexin-V and PI of 5 were added thereto, followed by incubation at room temperature for 15 minutes. Subsequently, after treating the lx binding complete solution of 400 to the EP-tube, the degree of apoptosis was quantitatively analyzed using flow cytometry.
  • a carrier RPMI 1640 medium, group 1
  • JO-58 MTD-EGFP protein group 2 fused JO-58 MTD to fluorescent protein EGFP
  • ⁇ 27 protein (group 3) unfused with MTD were 300 ⁇ Subcutaneously injected for 9 days. Tumor size of each group of mice was measured every day after protein treatment, and the results are shown in FIG. 15A.
  • tumor size of the cell-permeable p27 recombinant proteins HM, p27 and ⁇ 27 ⁇ , respectively, treated with the mice (groups 4 and 5) was higher than that of the control group (groups 1, 2 and 3). While growth was markedly inhibited, the weight of mice did not show significant changes in both control and cell-permeable p27 recombinant protein treatment groups.
  • Example ⁇ 8-1> In order to confirm the persistence of the anti-cancer effect in the body after the administration of the cell-permeable p27 recombinant protein ( ⁇ 27, ⁇ 27 ⁇ 0) according to the present invention, as shown in Example ⁇ 8-1>, two mice in each group were administered after 9 days of protein administration. Mice were selected to stop the carrier and all protein processing, and then tumor size was observed for 14 days.
  • HM lP 27 and HM lP 27Mi, 200 / zg cell-permeable p27 recombinant protein fused with JO-58 MTD and a control (RPMI 1640 medium, 200 ⁇ ) and Hp27 without MTD fused as a control.
  • a total of four groups were administered with protein (200 jug).
  • Each protein was intravenously injected for 14 days using a vernier caliper at a concentration of 1 / ⁇ when the tumor size (width 2 ⁇ length / 2) represented 50 Diiii.
  • mice were selected from each group after 14 days of protein administration as in Example ⁇ 9-1>. After stopping the carrier and all protein treatment, the tumor size was observed for 14 days.
  • the control group (carrier) showed a rapid increase in the size of the tumor, but in the cell-permeable p27 recombinant protein administration group (HM lP 27 and HM lP 27M0 similar without a sharp increase in tumor size) Levels were maintained according to the present invention.
  • the cell-permeable p27 recombinant protein is administered in vivo, indicating that the cell cycle of cancer cells continues to be fully reprogrammed and continues to act as a cell cycle inhibitor of cancer that normally normalizes cancer cells to prevent further cell division. .
  • Example 10 Analysis of Induction of Apoptosis After Administration of Cell Permeable p27 Recombinant Protein
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
  • mice were treated intravenously with intracellular injection of p27 recombinant proteins ( ⁇ 27 and HM lP 27M and the carrier and ⁇ 27 protein as a control group for 14 days, followed by a 14-day no-observation period. Then, the tumor tissues derived from each group of mice were separated and removed, and the extracted tumor tissues were fixed with formalin and washed, and then paraffin blocks were removed using paraffin dissolved at 62 ° C at an embedding center. The prepared paraffin block was cut to thickness 4 using a microtome, attached to a slide, and xylene was treated three times for 5 minutes to remove paraffin, followed by twice with 5 minutes of anhydrous ethanol.
  • p27 recombinant proteins ⁇ 27 and HM lP 27M and the carrier and ⁇ 27 protein
  • %, 80%, and 70% ethane were treated with 3 minutes each to hydrate and stored in PBS for 5 minutes, followed by 8 minutes in 0.1% sodium citrate solution.
  • Cells were permeabilized with dissolved 0.1% Triton® X-100 solution and washed twice with PBS for 2 minutes TUNEL reaction solution (50 ⁇ , Roche, USA) was treated on the tissue slides for 1 hour. After incubation in a 37 ° C. wet incubator, washed three times with PBS and observed with a fluorescence microscope. As a result, as shown in FIG.

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Abstract

L'invention concerne une protéine recombinante p27 à cellule perméable dont le domaine de transduction macromoléculaire (MTD) est fusionné avec le suppresseur de tumeur p27, un polynucléotide codant cette protéine, le vecteur d'expression de cette protéine, et une composition pharmaceutique destinée à des médicaments anticancéreux la contenant en tant que principe actif. La protéine recombinante p27 à cellule perméable de la présente invention transduit le suppresseur de tumeur p27 dans le noyau cellulaire et supprime la formation d'un complexe de kinase cycline-dépendante et de cycline, par revitalisation du mécanisme de transduction de signal qui participe à l'inhibition du cycle cellulaire et à l'induction de la mort cellulaire, ce qui permet à une composition contenant cette protéine en tant que principe actif d'être utilisée comme médicament anticancéreux dans la prévention et/ou le traitement de divers cancers.
PCT/KR2009/002588 2008-05-16 2009-05-15 Protéine recombinante p27 à cellule perméable, un polynucléotide codant cette protéine et une composition anticancéreuse la contenant en tant que principe actif WO2009139599A2 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8540988B2 (en) 2006-02-13 2013-09-24 Alethia Biotherapeutics Inc. Antibodies that bind polypeptides involved in the process of bone remodeling
US8741289B2 (en) 2009-10-06 2014-06-03 Alethia Biotherapeutics Inc. Siglec 15 antibodies in treating bone loss-related disease
WO2016028036A1 (fr) 2014-08-17 2016-02-25 Cellivery Therapeutics, Inc. Séquences de domaine de transduction macromoléculaire avancé (amtd) pour l'amélioration de la perméabilité cellulaire, polynucléotides codant pour celles-ci, procédé d'identification des caractéristiques uniques des amtd les comprenant, procédé de développement des séquences d'amtd les comprenant
US9493562B2 (en) 2012-07-19 2016-11-15 Alethia Biotherapeutics Inc. Anti-Siglec-15 antibodies
US10323072B2 (en) 2015-08-18 2019-06-18 Cellivery Therapeutics, Inc. Cell-permeable (CP)-Δ SOCS3 recombinant protein and uses thereof
US10385103B2 (en) 2014-08-27 2019-08-20 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US10508265B2 (en) 2015-08-10 2019-12-17 Cellivery Therapeutics, Inc. Cell-permeable reprogramming factor (iCP-RF) recombinant protein and use thereof
US10662419B2 (en) 2015-07-27 2020-05-26 Cellivery Therapeutics, Inc. Cell-permeable (ICP) parkin recombinant protein and use thereof
US10669531B2 (en) 2015-08-10 2020-06-02 Cellivery Therapeutics, Inc. Cell-permeable Cre (iCP-Cre) recombinant protein and use thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALAN HO ET AL.: 'Synthetic Protein Transduction Domains: Enhanced Transduction Potential in Vitro and in Vivo' CANCER RESEARCH vol. 61, 15 January 2001, pages 474 - 477 *
ERIC L. SNYDER ET AL.: 'Anti-cancer protein transduction strategies: reconstitution of p27 tumor suppressor function' JOURNAL OF CONTROLLED RELEASE vol. 91, 28 August 2003, pages 45 - 51 *
JEHANGIR S. WADIA ET AL.: 'Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer' ADVANCED DRUG DELIVERY REVIEWS vol. 57, no. 4, 28 February 2005, pages 579 - 596 *
LIM, SE JIN ET AL.: 'Protein transduction domain (PTD) and its application' BIOWAVE, [Online] vol. 8, no. 14, 2006, pages 1 - 17 Retrieved from the Internet: <URL:http://bric.postech.ac.kr/webzine> *

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US8540988B2 (en) 2006-02-13 2013-09-24 Alethia Biotherapeutics Inc. Antibodies that bind polypeptides involved in the process of bone remodeling
US9040246B2 (en) 2006-02-13 2015-05-26 Alethia Biotherapeutics Inc. Methods of making antibodies that bind polypeptides involved in the process of bone remodeling
US9067984B2 (en) 2006-02-13 2015-06-30 Alethia Biotherapeutics Inc. Methods of impairing osteoclast differentiation using antibodies that bind Siglec-15
US9695419B2 (en) 2006-02-13 2017-07-04 Daiichi Sankyo Company, Limited Polynucleotides and polypeptide sequences involved in the process of bone remodeling
US8741289B2 (en) 2009-10-06 2014-06-03 Alethia Biotherapeutics Inc. Siglec 15 antibodies in treating bone loss-related disease
US8900579B2 (en) 2009-10-06 2014-12-02 Alethia Biotherapuetics Inc. Siglec-15 antibodies in treating bone loss-related disease
USRE47672E1 (en) 2009-10-06 2019-10-29 Daiichi Sankyo Company, Limited Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
US9388242B2 (en) 2009-10-06 2016-07-12 Alethia Biotherapeutics Inc. Nucleic acids encoding anti-Siglec-15 antibodies
US9617337B2 (en) 2009-10-06 2017-04-11 Daiichi Sankyo Company, Limited Siglec-15 antibodies in treating bone loss-related disease
US9493562B2 (en) 2012-07-19 2016-11-15 Alethia Biotherapeutics Inc. Anti-Siglec-15 antibodies
US10323063B2 (en) 2014-08-17 2019-06-18 Cellivery Therapeutics, Inc. Advanced macromolecule transduction domain (aMTD) sequences for improvement of cell-permeability, polynucleotides encoding the same, method to identify the unique features of aMTDs comprising the same, method to develop the aMTD sequences comprising the same
WO2016028036A1 (fr) 2014-08-17 2016-02-25 Cellivery Therapeutics, Inc. Séquences de domaine de transduction macromoléculaire avancé (amtd) pour l'amélioration de la perméabilité cellulaire, polynucléotides codant pour celles-ci, procédé d'identification des caractéristiques uniques des amtd les comprenant, procédé de développement des séquences d'amtd les comprenant
JP2017527280A (ja) * 2014-08-17 2017-09-21 セリベリー セラピューティクス，インコーポレーテッド 細胞透過性の改善のための進化型巨大分子伝達ドメイン（ａｄｖａｎｃｅｄ ｍａｃｒｏｍｏｌｅｃｕｌｅ ｔｒａｎｓｄｕｃｔｉｏｎ ｄｏｍａｉｎ）（ＡＭＴＤ）配列、それをコードするポリヌクレオチド、それを含むＡＭＴＤの固有の特性を同定する方法、それを含むＡＭＴＤ配列を開発する方法
EP3180352A4 (fr) * 2014-08-17 2018-06-20 Cellivery Therapeutics Inc. Séquences de domaine de transduction macromoléculaire avancé (amtd) pour l'amélioration de la perméabilité cellulaire, polynucléotides codant pour celles-ci, procédé d'identification des caractéristiques uniques des amtd les comprenant, procédé de développement des séquences d'amtd les comprenant
US10774123B2 (en) 2014-08-27 2020-09-15 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (CP-BMP) recombinant protein and use thereof
US10385103B2 (en) 2014-08-27 2019-08-20 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US10781241B2 (en) 2014-08-27 2020-09-22 Cellivery Therapeutics, Inc. Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US10787492B2 (en) 2014-08-27 2020-09-29 Cellivery Therapeutics, Inc. Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US10961292B2 (en) 2014-08-27 2021-03-30 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US10975132B2 (en) 2014-08-27 2021-04-13 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US11279743B2 (en) 2014-08-27 2022-03-22 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (CPBMP) recombinant protein and use thereof
US10662419B2 (en) 2015-07-27 2020-05-26 Cellivery Therapeutics, Inc. Cell-permeable (ICP) parkin recombinant protein and use thereof
US10508265B2 (en) 2015-08-10 2019-12-17 Cellivery Therapeutics, Inc. Cell-permeable reprogramming factor (iCP-RF) recombinant protein and use thereof
US10669531B2 (en) 2015-08-10 2020-06-02 Cellivery Therapeutics, Inc. Cell-permeable Cre (iCP-Cre) recombinant protein and use thereof
US10689424B2 (en) 2015-08-18 2020-06-23 Cellivery Therapeutics, Inc. Cell-permeable (CP)-Δ SOCS3 recombinant protein and uses thereof
US10323072B2 (en) 2015-08-18 2019-06-18 Cellivery Therapeutics, Inc. Cell-permeable (CP)-Δ SOCS3 recombinant protein and uses thereof

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