WO2012165927A2 - Secretory fusion protein for target tracking - Google Patents
Secretory fusion protein for target tracking Download PDFInfo
- Publication number
- WO2012165927A2 WO2012165927A2 PCT/KR2012/004401 KR2012004401W WO2012165927A2 WO 2012165927 A2 WO2012165927 A2 WO 2012165927A2 KR 2012004401 W KR2012004401 W KR 2012004401W WO 2012165927 A2 WO2012165927 A2 WO 2012165927A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- fusion protein
- target tracking
- target
- seq
- Prior art date
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Classifications
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- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Definitions
- the present invention relates to secretory target tracking fusion proteins, more specifically
- a secretory target tracer fusion protein comprising (a) a peptide with extracellular secretory function, (b) a molecular image reporter or therapeutic peptide or protein, and (c) a peptide with specific binding ability to a target cell.
- a secretion-type target fusion protein comprising a secretory peptide, a molecular image reporter or a therapeutic peptide or protein, and a peptide having a specific binding ability to a target cell.
- the fusion protein specifically binds to a target cell. It was confirmed that this effectively imaged, the present invention was completed.
- Another object of the present invention to provide a molecular imaging diagnostic agent or a tumor cell, infection or inflammatory disease, ischemic disease treatment using the secreted target tracking fusion protein.
- the present invention provides a peptide having an extracellular secretion function (W molecular imaging reporter or therapeutic peptide or protein, and (c) a peptide having a specific binding capacity to target cells)
- a secreted target tracer fusion protein consisting of: The schematic diagram of the secreted target tracer fusion protein is shown in FIG.
- the fusion protein of the present invention is characterized by having multifunctionality.
- multi-function means that, for example, when the secretory target tracking fusion protein of the present invention is Glue-mCheny-RGD, Glue generates not only extracellular secretion but also a bioluminescence image signal, and mCherry fluoresces To generate a signal, and RGD tracks integrin If the function is modified to cyclic RGD, which is known to have better binding capacity, the fusion protein provides excellent target ability.
- the fusion protein has three functions, and when the mCherry portion of the fusion protein is replaced with TRAIL having a therapeutic effect, TRAIL has the effect of a cell therapy, and RGD is expressed in tumor cells or tumor vessels.
- the secreted target tracking fusion protein of the present invention has three or more multifunctionals.
- the peptide having an extracellular secretory function is secreted gaussia luciferase (Sec-Glue), secretory antibody, or signal recognition particle (signal recognition particle) Characterized in that it is a peptide having an extracellular secretion function selected from the group consisting of a peptide comprising a hydrophobic amino acid sequence recognized by, but is not limited thereto. More specifically, the peptide having the extracellular secretory function is preferably secreted type Gaussian luciferase (Sec-Glue) having the amino acid sequence of SEQ ID NO: 3.
- the peptide having an extracellular secretion function is to have the function of allowing extracellular secretion of a molecular image reporter or therapeutic peptide or protein and a peptide having a specific binding ability to a target cell in cells or bacteria.
- the molecular image reporter is a fluorescent protein, a bioluminescent protein, a photoacoustic protein, a radionuclide and a radio contrast agent, an ultrasound contrast agent or a protein to which an MR contrast agent binds.
- the therapeutic peptide or protein is TRAIL, apoptin, cytolysin (cytolysin), INF- ⁇ , FASL, TNF- ⁇ , cytokines (IL-2 or IL-18), blood vessels Anti-angiogenic peptides (thrombospondin, endostatin), ischemic peptides (pro— angiogenic peptide-vascular endothelial growth factor, hepatocyte growth factor, characterized in that it is a therapeutic peptide or protein selected from the group consisting of antimicrobial peptides such as angiopoietin, placental growth factor or defensin and ⁇ ]. It is not. More specifically, the molecular image reporter is preferably a fluorescent protein mCheny having an amino acid sequence of SEQ ID NO: 4, the therapeutic peptide is a TRAIL having an amino acid sequence of SEQ ID NO: 6 desirable.
- the molecular image reporter or therapeutic peptide is characterized in that the peptide having a binding capacity with the molecular image reporter or therapeutic material.
- the substance includes not only a molecular image reporter or therapeutic peptide or protein, but also a biotinylated material (biotinlylated polyamine dendrimer carrying drug, biotinlylated geldanamycin), a diagnostic compound biotinylated materiaKbiotinlyated lipids with Tc—99m HMPAO, Avidinlyated saporin or avidinylated micorbubble can also be used, which can be used for treatment and diagnosis.
- biotin acceptor peptide eg, GLNDIFEAQKIEWHE
- binding to a biotinylated material is possible, or streptavidin labeling binding may be used secondly after biotin binding.
- biotin or streptavidin can be labeled for diagnosis and treatment.
- the peptides having specific binding capacity to the target cells are RGD, NGR (tumor angiogenesis targeting), Apopep-1 (apoptosis targeting peptide), GPC-3 (glypican-3) target peptide (liver cancer, germ adenocarcinoma, black) GPC-3 tracking peptides overexpressed in species), IL-12 target peptide, aminopeptidase P target peptide, ILll- ⁇ target peptide, Nucleolin target peptide, tumor lymphatic A) a peptide selected from the group consisting of target peptides. It is not. Table 1 below shows peptide sequences having specific binding capacities to target cells [Liu et al., Pat. Anticancer. Drug.
- the peptide having a specific binding capacity to the target cell is preferably RGD having an amino acid sequence of SEQ ID NO: 5. More preferably, the RGD has a repeated sequence three times or an amino acid sequence of SEQ ID NO. 7 forming a cyclic RGD.
- the secretory target tracking fusion protein of the present invention is secreted extracellularly to track and bind to the target cells by a peptide having a specific binding ability to the target cells, and imaged by a molecular imaging reporter, or through a therapeutic peptide or protein Target cells can be treated by mechanisms that further accelerate cell death.
- the fusion protein of the present invention is a therapeutic peptide linkage site between the peptide or therapeutic peptide that causes extracellular secretion of the fusion protein, for example, between each protein or peptide constituting the fusion protein.
- a peptide sequence that can be cleaved by MMP-2 (Matrix Metalloproteinase-2) or MMP-9 may be further included at the linkage between the peptide and the cell tracer peptide.
- the peptide sequence that can be cleaved by MMP # 2 or MMP-9 is, for example, PLGLAG.
- the secretory target tracking fusion protein of the present invention is preferably a peptide having an extracellular secretory function of SEQ ID NO: 3, a molecular image reporter of SEQ ID NO: 4 or a therapeutic peptide or protein of SEQ ID NO: 6, and a target cell of SEQ ID NO: 5 It is characterized by consisting of a peptide having a specific binding capacity.
- the fusion protein of the present invention is characterized in that the ⁇ 3 target tracking fusion protein having the amino acid sequence of SEQ ID NO: 2.
- Integrins are heterodimers in which ⁇ and ⁇ subunits are covalently linked, and the ⁇ subfamily has been known as a major mediator of interstitial adhesions and may have other functions such as direct mediation of cell-cell adhesions.
- the ⁇ 2 subfamily found in white blood cells contains receptors that mediate cell-cell interactions.
- the ⁇ 3 subfamily contains platelet glycoprotein Ilb / IIIa complexes and vitronectin receptors, which may play a critical role in tumor invasion and development into malignancies [Albelda et al. al, Cancer. Res. 50: 6757-6764, 1990.
- the integrin ⁇ v ⁇ 3 has been reported to increase its expression upon neovascularization, which is essential for the growth of cancer cells, and has potential as a target for anticancer drug carriers.
- Tetrac 3,3'5,5'-tetraiodothyroacetic acid
- Integrin ⁇ 3 is not only expressed in neovascularization of the tumor site, but is also expressed in ischemic tissues such as myocardial infarction limb ischemia, myocardial remodeling, myocardial retinopathy and joint and skin inflammatory tissues after myocardial infarction. Integrin ⁇ ⁇ ⁇ 3 expression can also be used to diagnose / evaluate lesions / deliver therapeutics [Lee et al., J. Korean. Med. Assoc. 52 (2): 135-142 (2009); Zhou et al., Theranotics. 1: 58-82 (2011). According to another aspect of the present invention, there is provided a gene encoding the secreted target tracking fusion protein of the present invention.
- the gene is characterized in that it has a nucleotide sequence of SEQ ID NO: 1.
- the gene is characterized in that it further comprises an inducible promoter (inducible promoter).
- the secreted target tracking fusion protein of the present invention can be used to induce the fusion protein only when necessary using an inducible promoter.
- an inducible promoter for example, using a promoter capable of inducing or stopping expression by tetracycline, the expression can be controlled by administering or eliminating tetracycline only when the production of the secreted target tracer fusion protein is required.
- Tetrasa Tetracycline (Tet) inducible expression system Tet- off and Tet-on system.
- This inducible promoter system is considered to be very useful when applied to gene therapy techniques of the secreted target tracking fusion protein of the present invention in a transgenic animal model or cell therapy technology.
- the invention provides a recombinant vector comprising a gene encoding said secreted target trace fusion protein.
- the recombinant vector expressing the gene may use any expression vector capable of expressing the gene, and preferably, pcDNA3.1, pRSET (B) and pcDNA3.0 may be used. Since the pRSET (B) vector is expressed in JM109 bacteria and the pcDNA vector is expressed in mammalian cells, the pRSET (B) vector can be used to obtain a large amount of secreted target tracking fusion protein that can be traced to target cells.
- the recombinant vector is pcDNA3.0-sGluc-mCherry-RGDx3 or pcDNA3.0_sGluc—mCherry—cRGD vector having a cleavage map of FIG. 3.
- the present invention provides a cell line transformed with the recombinant vector.
- the cell line may be a mammalian cell, a bacteria or yeast, and more specifically, the cell line is characterized in that the CHO (chinese hamster ovary cell), but is not limited thereto.
- CHO chinese hamster ovary cell
- the present invention after confirming the mRNA expression of a target molecule such as ⁇ ⁇ ⁇ 3 in breast cancer cell line (MDM-MB-231), lung cancer cell line (A549), human synovial cell lines (2046 and 2047) and CHO, it does not express the ⁇ 3.
- CHO was used as the transforming cell line.
- the transformed cell line is characterized in that the CHO—sGluc-mCherry-RGDX3 or CHO-sGluc-mCherry-cRGD.
- the transformed cell line CHO-sGluc-mCherry-RGDX3 or CHO—sGluc—mCherry—cRGD is a recombinant vector produced in CHO cells, P cDNA3.0-sGluc-mCherry-RGD 3 or pcDNA3. 0-sGluc-mCherry-cRGD vector was prepared by incorporation using liposomes (lipofectamine).
- liposomes lipofectamine
- the transformed cell line may be transformed by protoplast, electroporation, or viral gene transfer.
- pcDNA3.0-sGluc-mCheny—RGDx3 Genes were transferred using liposomes and transformed cell lines were prepared by selecting the cells into which the genes were introduced using antibiotics and FACS sorter.
- a transgenic animal model transformed with the recombinant vector can be prepared.
- the transgenic animal model may produce a transgenic mouse using a mouse, and continuously monitor the expression level of the target cell by the secretory imaging reporter of the secreted target tracking fusion protein of the present invention expressed in the transgenic mouse.
- constructing a transgenic mouse expressing a fusion protein of an RGD peptide and a secretion imaging reporter that tracks ⁇ expressed in renal neovascularization enables continuous non-invasive imaging of neovascular sites. Screening for substances that inhibit development or tumor angiogenesis can be used as an animal model.
- the secretion imaging reporter secreted from the cells of the animal model is localized at the tumor site. Imaging is possible, and therapeutic agents can be screened to reduce neovascularization of tumor tissues.
- the present invention provides a molecular imaging diagnostic agent containing the secreted target tracking fusion protein as an active ingredient.
- the secretory target tracking fusion protein is characterized by having an amino acid sequence of SEQ ID NO: 2.
- the present invention provides a tumor cell therapeutic agent containing the secreted target tracking fusion protein as an active ingredient.
- the therapeutic peptide eg, TRAIL
- the secreted target tracking fusion protein is known to be effective for treating tumor cells, Argiris et al., Exp. Biol Med.
- the secreted target tracking fusion protein can be used as a tumor cell therapy.
- the present invention provides an agent for treating an infectious or inflammatory disease containing a secreted target tracking fusion protein according to the present invention as an active ingredient.
- Therapeutic peptides eg defensins, cathelicidin LL-37, MIP-3a / CCL20, buforin I or ubiquicidin
- the secreted target tracking fusion protein can be used as a therapeutic agent for infectious or inflammatory diseases.
- the present invention is the secreted target tracking fusion protein of the present invention as an active ingredient Provided is an ischemic disease treatment.
- the therapeutic peptide of the fusion protein eg, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), angiopoietin or PLGF (placental growth factor)
- VEGF vascular endothelial growth factor
- HGF hepatocyte growth factor
- PLGF placental growth factor
- the secreted target trace fusion protein can be used as a therapeutic agent for ischemic disease.
- Tumor cell therapy, infection or inflammatory disease treatment or ischemic disease treatment agent of the present invention can be prepared by a method known in the pharmaceutical art for use as a therapeutic agent, a pharmaceutically acceptable carrier, excipient, diluent, etc.
- the mixture may be prepared and used in the form of powder, granules, tablets, capsulants, or injections. They may also be administered orally (eg, intravenously, subcutaneously, intraperitoneally, or topically) or orally.
- the therapeutic agents of the invention are administered in a therapeutically effective amount.
- therapeutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and includes the age, sex, weight, health condition, It may be appropriately selected depending on the symptoms of the disease, the time of administration and the method of administration, preferably 0.01-100 mg per day of adult.
- secreted target tracking fusion protein consisting of a peptide having a can be prepared using bacteria or mammalian cells, the fusion protein can specifically bind to the target cell, can be confirmed by imaging the binding.
- the induction promoter can be used to express the fusion protein only when necessary.
- the present invention can search for a therapeutic agent that can treat a target cell using a therapeutic peptide or protein. Therefore, the secretory target tracking fusion protein of the present invention can be usefully used as a molecular imaging agent or a tumor cell therapeutic agent, and can be used to search for a therapeutic agent.
- ischemic diseases CAD and CAD
- retinopathy CAD and CAD
- inflammatory diseases including myocardial infarction and lower limb ischemia other than tumors. This is believed to be possible.
- Figure 1 shows a schematic of the secreted target trace fusion protein of the present invention.
- Figure 2 is a schematic diagram showing the construction of pcDNA3. SGlu-mCherry-RGD> ⁇ 3 and pcDNA3.0-sGluc-mCherry-cRGD recombinant vector.
- Figure 3 shows a cleavage map of the recombinant vector.
- Figure 5 is the result of measuring the protein expression in culture medium and cell lysate when incubating the fusion protein of the present invention with bacteria.
- FIG. 6 is a result of measuring the protein expression after transforming the fusion protein of the present invention to JM109 bacteria, after separating the fusion protein from bacterial cells.
- Figure 8 is the result of measuring the protein expression in culture medium and cell lysate when incubating the fusion protein of the present invention with mammalian cells (CHO).
- 9 is a result of measuring whether the fusion protein of the present invention specifically binds to ⁇ 3 in CHO and synovium cells using a luminescent enzyme (Galusssia luciferase).
- 10 is a fusion protein of the present invention only in synovium cells using a fluorescent protein (mCherry fluorescent protein) in a confocal microscope image by culturing together a mammalian cell secreting the sGluc—mCheny—RGDx3 fusion protein and a synovium cell expressing ⁇ 3. After a proof that specifically binding to the ⁇ ⁇ ⁇ 3 it is.
- FIG. 11 is a confocal microscopy image obtained by incubating sGluc—mCherry-RGD> ⁇ 3 fusion protein and a U87MG-EGFP cell expressing ⁇ 3 together with sGluc—mCheny-RGDx3 valent in U87MG-EGFP cells. It is an image showing the fluorescent signal attached.
- the sGluc-mCherry fusion protein without RGD has no fluorescence signal due to failure to attach.
- sGluc-mCherry-RGDx3 fusion protein from mammalian cells secreting sGluc-mCherry fusion protein, and then treated to U87MG-EGFP cells expressing ⁇ ⁇ ⁇ 3, fusion protein sGluc-mCheny secreted from mammalian cells -RGDx3 is attached to U87MG-EGFP cells and shows fluorescence signal.
- sGluc-mCherry—RGDx3 produced from JM109 bacteria.
- sGluc-mCheny was isolated and concentrated to treat U87MG-EGFP cells expressing ⁇ 3 and CHO-EGFP cells not expressing ⁇ 3.
- images treated with sGluc-mCherry-RGD 3 specific binding was observed in U87MG-EGFP cells expressing ⁇ 3, and specific binding was observed in both cells in (b) images treated with sGluc-mCheny. It doesn't work.
- PCR was used to amplify only the mCherry gene in the pmR—mCherry vector (Clonetech) and insert the amplified gene into the pRSET (B) vector to prepare a pRSET (B) -mCherry vector. Subsequently, pRSET (B) ⁇ mCherry-RGDx3 or pRSET (B) -mCheny—cRGD vector was prepared by inserting a previously prepared RGD sequence (RGDx3 or cRGD) into the prepared pRSET (B) _mCherry vector.
- PRSET (B) sGlu-mCherry-RGD> ⁇ 3 or pRSET (B) -sGlu-mCherry-cRGD vector was digested with BamH I and EcoRI restriction enzymes to sGluc ⁇ mCheny-RGDx3 or sGluc—mCheery-cRGD gene Then, it was inserted into the pcDNA3.0 vector (Invitrogen), and the final product, pcDNA3.0-sGlu-mCherry-RGD> ⁇ 3 or pcDNA3.0—sGlu ⁇ mCherry-cRGD vector was constructed.
- MDA—MB—231 breast cancer cell lines
- lung cancer cell lines A549
- human synovial cell lines (2046 and 2047
- Chinese hamster ovary cells CHO
- F MRNA expression was confirmed by PCR and DNA electrophoresis using primers (short strands complementary to specific sequences).
- the pRSET (B) vector Since the pRSET (B) vector is expressed in JM109 bacteria, it can be used to obtain a large amount of secreted target tracking fusion protein that can be traced to target cells.
- the pcDNA vector Since the pcDNA vector is expressed in mammalian cells, it can be used to obtain a large amount of secreted target tracking fusion protein capable of tracking target cells, and is also a vector that can be used for cell-based treatment.
- Bacteria that can produce the secreted target trace fusion protein of the present invention were prepared and incubated together, and then released into the culture medium.
- the pcDNA3.0—sGlu—mCheny-RGD> ⁇ 3 vector prepared in Example 1 was transformed into competent cells (DH5a), 50 yg / ml of ampicillin was added, and only 2 ml was taken at 37 °. Incubated overnight in C shaking incubator.
- the activity of the cell culture medium and the intracellular luciferase was 8 times higher in the luciferase activity in the cell culture medium than the intracellular luciferase activity.
- the fusion protein can be obtained in the same manner in culture and bacteria by introducing pRSET (B) -sGluc-mCheny RGDx3 or pRSET (B) -sGluc-mCherry-cRGD vector into JM109 bacteria.
- PRSET (B) -sGluc-mCheny—RGDX3 or pRSET (B) -sGluc-mCheny-cRGD were introduced into JM109 bacteria, respectively, to generate a large amount of fusion proteins, and the fusion protein was purified by a purification method using His—tag method. can do.
- sGluc-mCherry-RGDX3 fusion protein expressed in pRSET (B)-sGluc- mCherry- RGDX3 vector grafted sGluc-mCherry-RGDX3 fusion protein and pRSET (B) -sGluc-mCheny vector sGluc- mCherry fusion protein expressed After separation and purification, 0.5ug and lug were added to the 96-well plate, 100 ⁇ l of gaussia luciferase substrate was added, and luciferase activity was confirmed by IVIS image.
- Mammalian cells were produced that could produce the secreted target tracking fusion protein of the present invention and cultured together to confirm their release into the culture.
- the pcDNA3O-sGlu ⁇ mCherry-RGDx3 vector produced in Example 1 was applied to CHO cells without ⁇ 3 expression. Incorporation into cells using Lipofectamine, Invitrogen) Subsequently, subcultures were used to classify only cells into which PCDNA3.0—sGlu-mCheny-RGI 3 was safely introduced into cells using mCheny fluorescence with a flow cytometer (FIG. 7).
- the prepared CHO-sGluc-mCherry—RGC was stably inoculated with CHO cell line stably immobilized with CHO cell line and parental control CHO cells in a 24-well plate at 5xl0 4 / well. 24 hours after cell inoculation, the media of the wells were transferred to new tubes and the cells in the plates were washed twice with PBS. Then, 100 ul of cell lysis buffer (5 ⁇ lysis buffer, promega) was added to lyse the cells at the bottom of the plate. Lysed cells were placed in fresh E-tubes and centrifuged for 2 minutes at 1500 rpm.
- the culture medium obtained in the culture of CHO-sGluc-mCheny-RGD the next day was 2 ml / well on a plate to which the two types of cells were attached. Put in. After incubation for 48 hours, the cells were washed twice with PBS. Thereafter, PBS was added to each well at 1 ml / well, coelentrazine (0.25 ug in 50 ⁇ PBS) was added thereto, and an IVIS image was obtained (FIG. 9A).
- the reason for using luminescent enzyme is that it is difficult to image the signal of mCherry fluorescent protein with IVIS imaging equipment.
- luminase was more sensitive than fluorescent protein and luminase was used. In order to evaluate a video signal in a live animal, a high sensitivity video signal is required, but the sensitivity of the light signal is higher than that of mCherry.
- synovial cells 2052 that ⁇ 3 is expressed as embellish Nathan in Figure 8 showed the results that increasing the luminescence image signal of about two times compared to CHO not expressing ⁇ ⁇ ⁇ 3 cells (Fig. 9 ( ⁇ )). Therefore, it can be seen that the fusion protein is specifically bound to ⁇ ⁇ ⁇ 3.
- CHO, CHO—sGluc-mCheny—RGD and synovium cells (2052 cells) were inoculated on Nunc (Lab Tek-Chamber slide), and then 1> ⁇ 10 4 cells were added to CHO + synovium group and CH RGD + synovium group, respectively. Each well was inoculated by mixing. After 48 hours of incubation, the cells were washed twice with warm PBS. Then, 100 ⁇ of BD cytofix / cytoperm 1 ⁇ buffer was added to each well and fixed at 4 ° C. for 30 minutes. Then, 1 ⁇ Perm / wash buffer was added to 1 ml of BD perm / wash buffer and 9 ml of PBS in a 15 ml tube.
- U87MG cells U87MG-EGFP expressing enhanced GFP in the cytoplasm of U87MG cells expressing ⁇ 3 expressed in the cytoplasm and the fusion protein CHO—sGluc-mCherry-RGDx3 cells (sGluc—mCherry—RGD) of the present invention.
- ⁇ 3 secreting CHO cells were mixed in a number of 5> ⁇ 10 4 , ⁇ ⁇ ⁇ 5 , inoculated in an IWAKI Glass-based dish for 48 hours, and then observed by confocal microscopy. It was.
- the microscopic measurement results are shown in FIG. 11.
- the upper left image (A) was observed with GFP wavelength and U87MG-EGFP cells were observed.
- the upper right image (B) was observed with red wavelength to observe mCherry and CHO-sGluc— mCheny— RGDx3 cells.
- a red image signal is observed on the visual field center where U87MG-EGFP cells were observed at the GFP wavelength (bottom image (D)).
- U87MG cells (U87MG—EGFP) expressing enhanced GFP in the cytoplasm of U87MG cells expressing ⁇ 3 expressed in the cytoplasm (U87MG—EGFP) and fusion protein CHO-sGluc-mCherry cells (sGluc—mHO cells secreting CHO cells), respectively. > ⁇ 10 4 , ⁇ ⁇ ⁇ 5 were mixed and inoculated in an IWAKI Glass based dish, mixed incubation for 48 hours, and observed by confocal microscopy (Confocal microscopy).
- the microscopic measurement results are shown in FIG. 12.
- the upper left image (A) was observed with GFP wavelength, and U87MG-EGFP cells were observed, and the upper right image (B) was observed with red exaggeration to observe mCherry. can do.
- the red image signal was not observed in the middle of the field of view where cells were observed at the GFP wavelength (compare FIG. 10). It can be seen that the cells cannot be tracked.
- U87MG cells (U87MG—EGFP) expressing enhanced GFP in the cytoplasm of U87MG cells expressing ⁇ 3 expressed in the cytoplasm were inoculated in an IWAKI Glass based dish in a number of 5> ⁇ 10 4 , and sGluc- The culture solution was taken from a dish in which only mCherry—RGDx3 cells were cultured and placed in a dish inoculated with the U87MG cells, followed by culturing, followed by confocal microscopy. The microscopic measurement results are shown in FIG. 13.
- the upper left image (A) shows the U87MG—EGFP cells as a result of observation at the GFP wavelength
- the upper right image (B) shows the U87MG—EGFP cells at the GFP wavelength as the result of observing the mCherry.
- a red image signal is observed. This shows that the sGluc-mCherry-RGD 3 secreted target tracking fusion protein has the ability to track cells expressing ⁇ 3.
- U87MG cells expressing ⁇ 3 and CHO cells not expressing ⁇ 3 were transplanted into 5 ⁇ 10 6 cells in experimental animals, and after 2 weeks of implantation, 20ug injection of the fusion protein produced by bacteria through the tail vein. do.
- IVIS images were obtained by injecting luciferase substrate into the tail vein. to obtain an image signal only in U87MG cells which express the ⁇ 3 were This shows that the capability to keep track of cells expressing ⁇ ⁇ ⁇ 3 sGluc-mCheny- RGDx3 min-free target tracking fusion protein within the living body.
- a bacterial or mammalian cell comprising a secretory target tracking fusion protein consisting of an extracellular secretory function, a molecular image reporter or therapeutic peptide or protein, and a peptide having a specific binding ability to a target cell.
- the fusion protein can specifically bind to the target cell, can be confirmed by imaging the binding.
- the induction promoter can be used to express the fusion protein only when necessary.
- the present invention can search for a therapeutic agent that can treat a target cell using a therapeutic peptide or protein. Therefore, the secretory target tracking fusion protein of the present invention can be usefully used as a molecular imaging agent or a tumor cell therapeutic agent, and can be used to search for a therapeutic agent.
- Ischemic diseases including myocardial infarction and lower limb ischemia, and nephropathy, including inflammatory diseases, can be used as molecular imaging diagnostics and treatments for diseases related to the development and progression of the disease. This is believed to be possible.
Abstract
The present invention relates to a secretory fusion protein for target tracking, and more specifically, to a secretory fusion protein for target tracking comprising: a peptide with extracellular secretion; a molecular imaging reporter or a therapeutic peptide or protein; and a peptide which has a specific binding capacity to a target cell. According go the present invention, as the secretory fusion protein for target tracking can be prepared to comprise a peptide with extracellular secretion, a molecular imaging reporter or a therapeutic peptide or protein, and a peptide which has a specific binding capacity to a target cell, the fusion protein can specifically bind to a target cell, and binding occurrence can be checked by imaging. In addition, the fusion protein expression can be induced using an induction promoter, only when needed. Furthermore, it is possible to search for a therapeutic agent capable of treating the target cell using the therapeutic peptide or protein. Therefore, the secretory fusion protein for target tracking of the present invention is considered to be useful as a molecular imaging diagnostic agent or a tumor cell therapeutic agent.
Description
【명세서】 【Specification】
【발명의 명칭】 [Name of invention]
분비형 표적 추적 융합 단백질 Secretory Target Tracking Fusion Proteins
【기술분야】 Technical Field
본 발명은 분비형 표적 추적 융합 단백질에 관한 것으로， 더욱 구체적으로 The present invention relates to secretory target tracking fusion proteins, more specifically
(a) 세포외 분비기능을 갖는 펩타이드， (b) 분자영상 리포터 또는 치료용 펩타이드 또는 단백질， 및 (c) 표적세포에 특이적 결합능을 갖는 펩타이드로 이루어진 분비형 표적 추적 융합 단백질에 관한 것이다. A secretory target tracer fusion protein comprising (a) a peptide with extracellular secretory function, (b) a molecular image reporter or therapeutic peptide or protein, and (c) a peptide with specific binding ability to a target cell.
【배경기술】 Background Art
질환의 진단이나 치료에서 목적 물질이 선택적으로 표적 조직 또는 세포에만 머무르며 작용하도록 하기 위한 시도가 지속적으로 이루어져 왔다. 특히 종양의 치 료나 골관절염 및 뇌질환의 진단 및 치료영역에서 표적세포 또는 조직에 특이적으 로 존재하는 단백질 둥에 대한 많은 연구가 진행되어 왔으며， 이러한.물질들이 많이 도출되면서 이를 이용한 치료 연구도 활발히 진행되고 있다. 예컨대， 전립선암의 경 우 전립선특이항원 (Prostate Specific Antigen; PSA)이 많이 존재하고， 관절염이나 여러 종양조직에서는 기질금속단백질 분해효소 (Matrix Metalloprotease; MMP)가 정 상 조직에 비해 특이적으로 높게 발현하는 것을 확인하고， 이들은 질병 연구 및 치 료의 표적으로서 이용되고 있다. 그러나 질병의 진단 및 치료를 위해 사용하는 물질 이 이러한 표적에만 특이적으로 작용하지 않는 경우， 비특이적 분포에 의한 부작용 이나 낮은 영상도의 문제가 발생하므로, 표적에만 특이적으로 작용하는 제형의 개발 이 요구되고 있다. Attempts have been made in the diagnosis or treatment of diseases to ensure that the target substance selectively stays and acts only on the target tissue or cell. In particular, many studies have been conducted on the protein proteins that are specific to target cells or tissues in the areas of tumor treatment, osteoarthritis and brain diseases. It is becoming. For example, in prostate cancer, many prostate specific antigens (PSAs) are present. In arthritis and various tumor tissues, matrix metalloprotease (MMP) is specifically expressed higher than that of normal tissues. They are used as targets for disease research and treatment. However, if the materials used for the diagnosis and treatment of diseases do not act specifically on these targets, side effects due to non-specific distribution or problems of low image quality occur. Therefore, the development of formulations that act only on targets is required. It is becoming.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의
연구 노력한 결과, 기존의 질병 진단 또는 치료용 물질의 비 특이적 분포에 따른 부 작용 또는 낮은 영상도의 문제점을 최소화하고， 진단 또는 질병 치료용 물질을 표적 세포에만 특이적으로 전달시키기 위해， 세포외 분비기능을 갖는 펩타이드， 분자영상 리포터 또는 치료용 펩타이드 또는 단백질 및 표적세포에 특이적 결합능을 갖는 펩 타이드로 이루어진 분비형 표적 융합 단백질을 제조하였으며， 상기 융합 단백질이 표적세포에 특이적으로 결합하고， 이를 효과적으로 영상화함을 확인하고， 본 발명을 완성하게 되었다. Accordingly, the present inventors have diligence to overcome the problems of the prior art. As a result of our research, in order to minimize the problems of side effects or low image quality due to the non-specific distribution of the existing disease diagnosis or therapeutic substance, and to specifically deliver the diagnostic or disease therapeutic substance to the target cell, extracellular A secretion-type target fusion protein was prepared comprising a secretory peptide, a molecular image reporter or a therapeutic peptide or protein, and a peptide having a specific binding ability to a target cell. The fusion protein specifically binds to a target cell. It was confirmed that this effectively imaged, the present invention was completed.
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】 [Technical problem]
따라서 본 발명의 주된 목적은 세포외 분비 기능， 영상화 또는 치료 기능 및 표적 기능을 갖는 분비형 표적 추적 융합 단백질을 제공하는데 있다. It is therefore a primary object of the present invention to provide a secreted target tracking fusion protein with extracellular secretory function, imaging or therapeutic function and target function.
본 발명의 다른 목적은 상기 분비형 표적 추적 융합 단백질을 이용한 분자영상 진단제 또는 종양 세포， 감염 또는 염증 질환， 허혈 질환 치료제를 제공하는데 있다. Another object of the present invention to provide a molecular imaging diagnostic agent or a tumor cell, infection or inflammatory disease, ischemic disease treatment using the secreted target tracking fusion protein.
【기술적 해결방법】 Technical Solution
본 발명의 한 양태에 따르면， 본 발명은 (a) 세포외 분비기능을 갖는 펩타이 드， (W 분자영상 리포터 또는 치료용 펩타이드 또는 단백질， 및 (c) 표적세포에 특 이적 결합능을 갖는 펩타이드로 이루어진 분비 형 표적 추적 융합 단백질을 제공한 다. 상기 분비 형 표적 추적 융합 단백질의 모식도는 도 1에 나타내었다. According to an aspect of the present invention, the present invention provides a peptide having an extracellular secretion function (W molecular imaging reporter or therapeutic peptide or protein, and (c) a peptide having a specific binding capacity to target cells) Provided is a secreted target tracer fusion protein consisting of: The schematic diagram of the secreted target tracer fusion protein is shown in FIG.
본 발명의 융합 단백질은 다기능을 갖는 것을 특징으로 한다. 상기 용어 "다 기능"은 예컨대， 본 발명의 분비 형 표적 추적 융합 단백질이 Glue— mCheny-RGD인 경우， Glue가 세포외 분비기능뿐만 아니라 생물발광 (bioluminescence) 영상신호를 발 생시키고， mCherry는 형광신호를 발생시키고， RGD는 인테그린 (integrin)을 추적하는
기능을 나타내고， 결합능이 좀 더 우수함이 알려진 cyclic 형태의 RGD로 변형을 시 킨다면， 상기 융합 단백의 우수한 표적능을 제공한다. 이 융합 단백질은 상기 세 가 지의 다기능을 가지며, 또한 상기 융합 단백질의 mCherry 부분을 치료효과를 갖는 TRAIL로 치환시키게 되면, TRAIL은 세포치료제의 효과를 나타내며， 이때 RGD는 종양세포 또는 종양혈관에 발현되는 인테그린 (integrin)을 추적하는 기능을 가지게 되 므로， 본 발명의 분비형 표적 추적 융합 단백질은 3 가지 이상의 다기능을 갖는다. 본 발명의 융합 단백질에 있어서， 상기 세포외 분비기능을 갖는 펩타이드는 분비형 가우시아 루시퍼라제 (secretory gaussia luciferase; Sec-Glue), 분비형 항체 (secretory antibody), 또는 신호 인식 입자 (signal recognition particle)에 의해 인식 되는 소수성 아미노산 서열을 포함하는 펩타이드로 구성된 군에서 선택된 세포외 분비기능 갖는 펩타이드인 것을 특징으로 하며, 이에 한정되는 것은 아니다. 보다 구체적으로, 상기 세포외 분비기능을 갖는 펩타이드는 서열번호 3의 아미노산 서열 을 갖는 분비형 가우시아 루시퍼라제 (Sec-Glue)인 것이 바람직하다. The fusion protein of the present invention is characterized by having multifunctionality. The term "multi-function" means that, for example, when the secretory target tracking fusion protein of the present invention is Glue-mCheny-RGD, Glue generates not only extracellular secretion but also a bioluminescence image signal, and mCherry fluoresces To generate a signal, and RGD tracks integrin If the function is modified to cyclic RGD, which is known to have better binding capacity, the fusion protein provides excellent target ability. The fusion protein has three functions, and when the mCherry portion of the fusion protein is replaced with TRAIL having a therapeutic effect, TRAIL has the effect of a cell therapy, and RGD is expressed in tumor cells or tumor vessels. Since it has the function of tracking integrin, the secreted target tracking fusion protein of the present invention has three or more multifunctionals. In the fusion protein of the present invention, the peptide having an extracellular secretory function is secreted gaussia luciferase (Sec-Glue), secretory antibody, or signal recognition particle (signal recognition particle) Characterized in that it is a peptide having an extracellular secretion function selected from the group consisting of a peptide comprising a hydrophobic amino acid sequence recognized by, but is not limited thereto. More specifically, the peptide having the extracellular secretory function is preferably secreted type Gaussian luciferase (Sec-Glue) having the amino acid sequence of SEQ ID NO: 3.
상기 세포외 분비기능을 갖는 펩타이드는 세포 또는 박테리아 내에서 분자영 상 리포터 또는 치료용 펩타이드 또는 단백질 및 표적세포에 특이적 결합능을 갖는 펩타이드를 세포외로 분비되도록 하는 기능을 갖는 것이다. The peptide having an extracellular secretion function is to have the function of allowing extracellular secretion of a molecular image reporter or therapeutic peptide or protein and a peptide having a specific binding ability to a target cell in cells or bacteria.
본 발명의 융합 단백질에 있어서， 상기 분자영상 리포터는 형광 단백질 (fluorescent protein), 발광 단백질 (bioluminescent protein), 광음영상 단백질 (photoacoustic protein), 방사성핵종 및 방사선조영제， 초음파조영제 또는 MR 조영 제가 결합하는 단백질로 구성된 군에서 선택된 분자영상 리포터이며， 상기 치료용 펩타이드 또는 단백질은 TRAIL, apoptin, 사이토라이신 (cytolysin), INF-β, FASL, TNF-α, 사이토카인 (IL— 2 또는 IL-18), 혈관신생억제 펩타이드 (anti-angiogenic peptide - thrombospondin, endostatin), 허혈치료 펩타이드 (pro— angiogenic peptide- vascular endothelial growth factor, hepatocyte growth factor,
angiopoietin, placental growth factor) 또는 디 펜신 (defensin) 및 ^]·텔리 시 딘 (cathelicidin)과 같은 항균성 (antimicrobial) 펩타이드들로 구성 된 군에서 선택된 치 료용 펩타이드 또는 단백질인 것을 특징으로 하며， 이 에 한정 되 는 것은 아니다. 보 다 구체적 으로， 상기 분자영상 리포터는 서 열번호 4의 아미노산 서 열을 갖는 형광 단백 질 mCheny인 것 이 바람직 하며， 치료용 펩타이 드는 서 열번호 6의 아미 노산 서 열을 갖는 TRAIL인 것 이 바람직 하다. In the fusion protein of the present invention, the molecular image reporter is a fluorescent protein, a bioluminescent protein, a photoacoustic protein, a radionuclide and a radio contrast agent, an ultrasound contrast agent or a protein to which an MR contrast agent binds. Molecular imaging reporter selected from the group consisting of, the therapeutic peptide or protein is TRAIL, apoptin, cytolysin (cytolysin), INF-β, FASL, TNF-α, cytokines (IL-2 or IL-18), blood vessels Anti-angiogenic peptides (thrombospondin, endostatin), ischemic peptides (pro— angiogenic peptide-vascular endothelial growth factor, hepatocyte growth factor, characterized in that it is a therapeutic peptide or protein selected from the group consisting of antimicrobial peptides such as angiopoietin, placental growth factor or defensin and ^]. It is not. More specifically, the molecular image reporter is preferably a fluorescent protein mCheny having an amino acid sequence of SEQ ID NO: 4, the therapeutic peptide is a TRAIL having an amino acid sequence of SEQ ID NO: 6 desirable.
또한， 상기 분자영상 리포터 또는 치료용 펩타이드는 분자영상 리포터 또는 치료용 물질과 결합능을 갖는 펩타이드인 것을 특징으로 한다. 상기 물질은 분자영 상 리포터 또는 치료용 펩타이드 또는 단백 질뿐만 아니 라， 치 료용 화합물인 biotinylated material (biotinlylated polyamine dendrimer carrying drug, biotinlylated geldanamycin), 진단용 화합물인 biotinylated materiaKbiotinlyated lipids with Tc— 99m HMPAO)이 있으며， avidinlyated saporin 혹은 avidinylated micorbubble 둥도 될 수 있으며， 이들은 치료 및 진단용으로 이용될 수 있다. In addition, the molecular image reporter or therapeutic peptide is characterized in that the peptide having a binding capacity with the molecular image reporter or therapeutic material. The substance includes not only a molecular image reporter or therapeutic peptide or protein, but also a biotinylated material (biotinlylated polyamine dendrimer carrying drug, biotinlylated geldanamycin), a diagnostic compound biotinylated materiaKbiotinlyated lipids with Tc—99m HMPAO, Avidinlyated saporin or avidinylated micorbubble can also be used, which can be used for treatment and diagnosis.
예컨대， biotin acceptor peptide (예컨대， GLNDIFEAQKIEWHE)를 본 발명 의 융합 단백질에 도입시 키 면， biotinylated material과의 결합이 가능하고， 또는 biotin 결합 후 2차적으로 streptavidin 표지 물질 결합을 이용할 수 있다. 즉, biotin 또는 streptavidin에 진단 및 치 료용 물질을 표지 하면 진단 및 치 료에 이용이 가능하다. 또한， 상기 표적세포에 특이 적 결합능을 갖는 펩타이드는 RGD, NGR (종양혈 관신생 targeting), Apopep-1 (apoptosis targeting peptide), GPC-3(glypican-3) 표적 펩타이드 (간암， 생식 선암, 흑색종 등에서 과발현되는 GPC-3 추적 펩타이드)， IL-12 표적 펩타이 드， 아미 노펩티다아제 (aminopeptidase) P 표적 펩타이드， ILll-γ 표적 펩 타이드， 뉴클레오린 (Nucleolin) 표적 펩타이드， 종양 림프절 (tumor lymphatic) 표적 펩타이드로 구성 된 군에서 선택된 펩타이 드인 것을 특징으로 하며， 이 에 한정 되 는
것은 아니다. 하기 표 1에 표적세포에 특이적 결합능을 갖는 펩타이드 서열에 대해 나타내었다 [Liu et al., Pat. Anticancer. Drug. Discov. Nov;3(3):202— 8， 2008]. 보다 구체적으로, 상기 표적세포에 특이적 결합능을 갖는 펩타이드는 서열번호 5의 아미 노산 서열을 갖는 RGD인 것이 바람직하다. 더욱 바람직하게는 상기 RGD는 3번 반 복된 연속서열을 가지거나, cyclic 형태의 RGD를 형성하는 서열번호 7의 아미노산 서열을 갖는 것이 좋다. For example, when a biotin acceptor peptide (eg, GLNDIFEAQKIEWHE) is introduced into the fusion protein of the present invention, binding to a biotinylated material is possible, or streptavidin labeling binding may be used secondly after biotin binding. In other words, biotin or streptavidin can be labeled for diagnosis and treatment. In addition, the peptides having specific binding capacity to the target cells are RGD, NGR (tumor angiogenesis targeting), Apopep-1 (apoptosis targeting peptide), GPC-3 (glypican-3) target peptide (liver cancer, germ adenocarcinoma, black) GPC-3 tracking peptides overexpressed in species), IL-12 target peptide, aminopeptidase P target peptide, ILll-γ target peptide, Nucleolin target peptide, tumor lymphatic A) a peptide selected from the group consisting of target peptides. It is not. Table 1 below shows peptide sequences having specific binding capacities to target cells [Liu et al., Pat. Anticancer. Drug. Discov. Nov; 3 (3): 202— 8, 2008]. More specifically, the peptide having a specific binding capacity to the target cell is preferably RGD having an amino acid sequence of SEQ ID NO: 5. More preferably, the RGD has a repeated sequence three times or an amino acid sequence of SEQ ID NO. 7 forming a cyclic RGD.
[표 1. 표적세포에 특이적 결합능을 갖는 펩타이드] Table 1. Peptides with Specific Binding Ability to Target Cells]
본 발명의 분비형 표적 추적 융합 단백질은 세포외로 분비되어 표적세포에 특이적 결합능을 갖는 펩타이드에 의해 표적세포를 추적하여 이와 결합하고, 이를 분자영상 리포터에 의해 영상화 하거나， 치료용 펩타이드 또는 단백질을 통해 세포 사멸이 더 가속화되는 기전 등으로 표적세포를 치료할 수 있다.
본 발명의 융합 단백질은 특정 단백질 또는 펩타이드 부위를 종양부위에서 유리되게 하기 위해， 융합 단백질을 구성하는 각 단백질 또는 펩타이드 사이 예컨대, 융합 단백질의 세포외 분비를 일으키는 펩타이드와 치료용 펩타이드 연결부위， 치료 용 펩타이드와 세포 추적 펩타이드의 연결부위에 MMP— 2(Matrix Metalloproteinase-2) 또는 MMP-9에 의해 절단될 수 있는 펩타이드 서열이 더 포 함될 수 있다. 상기 MMPᅳ 2 또는 MMP-9에 의해 절단될 수 있는 펩타이드 서열은 예컨대， PLGLAG가 바람직하다. 본 발명의 융합 단백질에 상기 MMP에 의해 절단 될 수 있는 펩타이드 서열을 더 포함시킴으로써 TRAIL과 같은 세포 치료 효과를 갖는 펩타이드를 종양부위에서만 분리될 수 있도록 할 수 있다. 본 발명의 분비형 표적 추적 융합 단백질은 바람직하게， 서열번호 3의 세포 외 분비기능을 갖는 펩타이드, 서열번호 4의 분자영상 리포터 또는 서열번호 6의 치 료용 펩타이드 또는 단백질 및 서열번호 5의 표적세포에 특이적 결합능을 갖는 펩 타이드로 구성되는 것을 특징으로 한다. The secretory target tracking fusion protein of the present invention is secreted extracellularly to track and bind to the target cells by a peptide having a specific binding ability to the target cells, and imaged by a molecular imaging reporter, or through a therapeutic peptide or protein Target cells can be treated by mechanisms that further accelerate cell death. In order to release a specific protein or peptide site from the tumor site, the fusion protein of the present invention is a therapeutic peptide linkage site between the peptide or therapeutic peptide that causes extracellular secretion of the fusion protein, for example, between each protein or peptide constituting the fusion protein. A peptide sequence that can be cleaved by MMP-2 (Matrix Metalloproteinase-2) or MMP-9 may be further included at the linkage between the peptide and the cell tracer peptide. The peptide sequence that can be cleaved by MMP # 2 or MMP-9 is, for example, PLGLAG. By further including a peptide sequence that can be cleaved by the MMP in the fusion protein of the present invention, it is possible to separate the peptide having a cell therapeutic effect such as TRAIL only at the tumor site. The secretory target tracking fusion protein of the present invention is preferably a peptide having an extracellular secretory function of SEQ ID NO: 3, a molecular image reporter of SEQ ID NO: 4 or a therapeutic peptide or protein of SEQ ID NO: 6, and a target cell of SEQ ID NO: 5 It is characterized by consisting of a peptide having a specific binding capacity.
본 발명의 융합 단백질은 서열번호 2의 아미노산 서열을 갖는 ανβ3 표적 추 적 융합 단백질인 것을 특징으로 한다. The fusion protein of the present invention is characterized in that the ανβ3 target tracking fusion protein having the amino acid sequence of SEQ ID NO: 2.
인테그린은 α, β 서브유닛이 비공유결합으로 연결된 헤테로다이머이며， βΐ 서브 패밀리는 세포간질 유착의 주요 매개인자로 알려져 왔으며， 다른 기능들 예컨 대, 세포 -세포 유착의 직접적인 매개와 같은 기능을 가지고 있을 가능성이 있다는 연구보고가 있다 [Larjava et al, J. Cell. Biol. 110:803-815， 1990]. 백혈구에서 발견 되는 β2 서브패밀리는 세포—세포 상호작용을 매개하는 수용체를 포함한다. 그리고 β3 서브패밀리는 혈소판 당단백질인 Ilb/IIIa 복합체와 비트로넥틴 (vitronectin) 수용 체를 포함하며， 종양의 침윤성과 악성종양으로의 발달에 있어 증요한 역할을 할 가 능성을 내포하고 있다 [Albelda et al, Cancer. Res. 50:6757-6764， 1990].
상기 인테그린 ανβ3은 암세포의 성장에 필수적인 신생혈관 생성시에 그 발 현이 증가함이 보고되어 있어, 항암제 전달체의 표적으로써의 가능성이 있다. 인테 그린 수용체와 리간드의 상호작용 예컨대， 인테그린 ανβ3과 그 리간드의 상호작용 을 차단하는 3,3'5,5'-tetraiodothyroacetic acid (Tetrac)은 인테그린 ανβ3 길항물질 로서 인테그린에 의한 종양 세포의 확산을 방지한다. 또한 암세포 표면에 발현하는 인테그린의 신호전달을 억제함으로써 세포 사멸을 유도하여 암세포의 사멸을 일으 킨다 [Yalcin et al. Anticancer. Res. 10， 3825-31, 2009]. Integrins are heterodimers in which α and β subunits are covalently linked, and the βΐ subfamily has been known as a major mediator of interstitial adhesions and may have other functions such as direct mediation of cell-cell adhesions. There are reports of the possibility [Larjava et al, J. Cell. Biol. 110: 803-815, 1990. The β2 subfamily found in white blood cells contains receptors that mediate cell-cell interactions. The β3 subfamily contains platelet glycoprotein Ilb / IIIa complexes and vitronectin receptors, which may play a critical role in tumor invasion and development into malignancies [Albelda et al. al, Cancer. Res. 50: 6757-6764, 1990. The integrin αvβ3 has been reported to increase its expression upon neovascularization, which is essential for the growth of cancer cells, and has potential as a target for anticancer drug carriers. Interactions of integrin receptors with ligands such as 3,3'5,5'-tetraiodothyroacetic acid (Tetrac), which blocks the interaction of integrin α ν β3 with its ligands, is an integrin ανβ3 antagonist and the proliferation of tumor cells by integrin To prevent. In addition, by inhibiting the signaling of integrins expressed on the surface of cancer cells, cell death is induced, causing cancer cell death [Yalcin et al. Anticancer. Res. 10, 3825-31, 2009].
인테그린 ανβ3은 종양 부위의 신생혈관에 발현될 뿐만 아니라， 심근경색 사 지 허혈 둥과 같은 허혈조직， 심근경색 후 심근재구성 (remodeling), 안구 망막증 및 관절 및 피부 염증조직에도 과 발현되므로 허혈조직의 이러한 병소의 진단 /상태 평 가 / 및 치료제 전달에도 인테그린 ανβ3 발현을 이용할 수 있다 [Lee et al., J. Korean. Med. Assoc. 52(2)： 135 - 142(2009)； Zhou et al., Theranotics. 1:58-82(2011)]. 본 발명의 다른 양태에 따르면， 본 발명의 상기 분비형 표적 추적 융합 단백 질을 코딩하는 유전자를 제공한다. Integrin ανβ3 is not only expressed in neovascularization of the tumor site, but is also expressed in ischemic tissues such as myocardial infarction limb ischemia, myocardial remodeling, myocardial retinopathy and joint and skin inflammatory tissues after myocardial infarction. Integrin α ν β3 expression can also be used to diagnose / evaluate lesions / deliver therapeutics [Lee et al., J. Korean. Med. Assoc. 52 (2): 135-142 (2009); Zhou et al., Theranotics. 1: 58-82 (2011). According to another aspect of the present invention, there is provided a gene encoding the secreted target tracking fusion protein of the present invention.
본 발명에 있어서， 상기 유전자는 서열번호 1의 염기서열을 갖는 것을 특징 으로 한다. In the present invention, the gene is characterized in that it has a nucleotide sequence of SEQ ID NO: 1.
상기 유전자는 유도형 촉진자 (inducible promoter)를 더 포함하는 것을 특징 으로 한다. 본 발명의 분비형 표적 추적 융합 단백질을 유도형 촉진자 (inducible promoter)를 이용하여， 필요시에만 상기 융합 단백질이 발현되도록 할 수 있다. 예컨 대， 테트라사이클린 (tetracycline)에 의해 발현을 유도하거나 중지할 수 있는 촉진자 를 이용하면， 분비형 표적 추적 융합 단백질 생산이 필요한 시기에만 테트라사이클 린 (tetracycline)을 투여하거나 제거함으로써 발현을 조절할 수 있게 된다 [테트라사
이클린 발현유도 체계 (Tetracycline(Tet) inducible expression system; Tet— off and Tet-on system)]. 이러한 유도형 촉진자 체계는 형질전환 동물모델 혹은 세포치료기 술에서 본 발명의 분비형 표적 추적 융합 단백질을 유전자치료 기법에 적용하는 경 우 매우 유용하게 사용이 가능할 것으로 사료된다. 본 발명의 다른 양태에 따르면， 본 발명은 상기 분비형 표적 추적 융합 단백 질을 코딩하는 유전자를 포함하는 재조합 백터를 제공한다. The gene is characterized in that it further comprises an inducible promoter (inducible promoter). The secreted target tracking fusion protein of the present invention can be used to induce the fusion protein only when necessary using an inducible promoter. For example, using a promoter capable of inducing or stopping expression by tetracycline, the expression can be controlled by administering or eliminating tetracycline only when the production of the secreted target tracer fusion protein is required. Tetrasa Tetracycline (Tet) inducible expression system; Tet- off and Tet-on system. This inducible promoter system is considered to be very useful when applied to gene therapy techniques of the secreted target tracking fusion protein of the present invention in a transgenic animal model or cell therapy technology. According to another aspect of the invention, the invention provides a recombinant vector comprising a gene encoding said secreted target trace fusion protein.
본 발명에 있어서， 상기 유전자를 발현하는 재조합 백터는 상기 유전자를 발 현할 수 있는 어떠한 발현백터도 이용될 수 있으며， 바람직하게는 pcDNA3.1, pRSET(B) 및 pcDNA3.0을 사용할 수 있다. 상기 pRSET(B) 백터는 JM109 박테리 아에서 단백질 발현이 되고， pcDNA 백터는 포유동물세포에서 단백질 발현이 되므 로， 이를 이용하면 표적세포 추적 가능한 다량의 분비형 표적 추적 융합 단백질을 얻을 수 있다. In the present invention, the recombinant vector expressing the gene may use any expression vector capable of expressing the gene, and preferably, pcDNA3.1, pRSET (B) and pcDNA3.0 may be used. Since the pRSET (B) vector is expressed in JM109 bacteria and the pcDNA vector is expressed in mammalian cells, the pRSET (B) vector can be used to obtain a large amount of secreted target tracking fusion protein that can be traced to target cells.
본 발명에 있어서， 상기 재조합 백터는 도 3의 개열지도를 갖는 pcDNA3.0-sGluc-mCherry-RGDx3 또는 pcDNA3.0_sGluc— mCherry—cRGD 백터인 것을 특징으로 한다. 본 발명의 다른 양태에 따르면， 본 발명은 상기 재조합 백터로 형질 전환된 세포주를 제공한다. In the present invention, the recombinant vector is pcDNA3.0-sGluc-mCherry-RGDx3 or pcDNA3.0_sGluc—mCherry—cRGD vector having a cleavage map of FIG. 3. According to another aspect of the present invention, the present invention provides a cell line transformed with the recombinant vector.
본 발명에 있어서， 상기 세포주는 포유세포， 박테리아 또는 이스트일 수 있으 며, 보다 구체적으로 상기 세포주는 CHO(chinese hamster ovary cell)인 것을 특징 으로 하며， 이에 한정되는 것은 아니다. 본 발명에서는 유방암 세포주 (MDM-MB-231), 폐암 세포주 (A549), 사람활막세포주 (2046 및 2047) 및 CHO에서 표적 분자 예컨대， ανβ3의 mRNA 발현을 확인한 후， 상기 ανβ3을 발현하지 않는
CHO를 형질전환 세포주로 이용하였다. In the present invention, the cell line may be a mammalian cell, a bacteria or yeast, and more specifically, the cell line is characterized in that the CHO (chinese hamster ovary cell), but is not limited thereto. In the present invention, after confirming the mRNA expression of a target molecule such as α ν β3 in breast cancer cell line (MDM-MB-231), lung cancer cell line (A549), human synovial cell lines (2046 and 2047) and CHO, it does not express the ανβ3. CHO was used as the transforming cell line.
상기 형질 전환된 세포주는 CHO— sGluc-mCherry-RGDX3 또는 CHO-sGluc-mCherry-cRGD인 것을 특징으로 한다. The transformed cell line is characterized in that the CHO—sGluc-mCherry-RGDX3 or CHO-sGluc-mCherry-cRGD.
본 발명의 구체적인 실시예에서， 형질전환된 세포주 CHO-sGluc-mCherry-RGDX3 또는 CHO— sGluc— mCherry— cRGD는 CHO 세포에 제 작된 재조합 백터인 PcDNA3.0-sGluc-mCherry-RGD 3 또는 pcDNA3.0-sGluc-mCherry-cRGD 백터를 리포좀 (lipofectamine)을 이용하여 이입시 킴으로써 제작하였다. 또한, 상기 형질전환된 세포주와 세포를 배양하는 경우， 배양 배지 및 세포 용해물에서 본 발명의 융합 단백질이 발현되는 것을 확인하였다 (실시 예 3-2 및 도 7 참조). In a specific embodiment of the present invention, the transformed cell line CHO-sGluc-mCherry-RGDX3 or CHO—sGluc—mCherry—cRGD is a recombinant vector produced in CHO cells, P cDNA3.0-sGluc-mCherry-RGD 3 or pcDNA3. 0-sGluc-mCherry-cRGD vector was prepared by incorporation using liposomes (lipofectamine). In addition, when culturing the transformed cell line and the cell, it was confirmed that the fusion protein of the present invention is expressed in the culture medium and cell lysate (see Example 3-2 and Figure 7).
본 발명에 있어서， 상기 형질전환된 세포주의 형질전환방법은 원형질체법 (protoplast), 전기천 공법 (dectroporation) 또는 바이러스 유전자 전달법 등을 사용할 수 있으나, 본 발명에서는 pcDNA3.0-sGluc-mCheny— RGDx3유전자를 리포좀을 이 용하여 이입시키고， 이 유전자가 이입된 세포를 항생제 및 FACS sorter를 이용하여 선택해냄으로써 형질 전환된 세포주를 제작하였다. In the present invention, the transformed cell line may be transformed by protoplast, electroporation, or viral gene transfer. In the present invention, pcDNA3.0-sGluc-mCheny—RGDx3 Genes were transferred using liposomes and transformed cell lines were prepared by selecting the cells into which the genes were introduced using antibiotics and FACS sorter.
또한， 상기 재조합 백터로 형질 전환된 형질전환 동물모델을 제작할 수 있다. 상기 형질전환 동물모델은 마우스를 이용하여 형질전환 마우스를 제작할 수 있으며， 상기 형질전환 마우스에서 발현된 본 발명의 분비형 표적 추적 융합 단백질의 분비 영상 리포터에 의해 지속적으로 표적세포의 발현정도를 관찰할 수 있다. 예컨대， 신 생혈관에 발현되는 ανβ를 추적하는 RGD 펩타이드와 분비영상 리포터의 융합 단백 질을 발현하는 형질전환 마우스를 구축하면， 지속적으로 신생혈관부위를 비침습적으 로 영상이 가능하게 되며， 신생혈관발생 또는 종양혈관발생을 저해하는 물질의 검색 동물모델로 사용이 가능하다. 상기 동물모델에 종양을 이식하여 신생혈관이 발생하 면， 이 종양에 동물모델의 세포에서 분비된 분비영상 리포터가 종양부위에 국소화되
어 영상화가 가능하며， 종양조직의 신생혈관발생을 감소시키는 치료 물질을 스크리 닝할 수 있다. 본 발명의 한 양태에 따르면， 본 발명은 상기 분비형 표적 추적 융합 단백질 을 유효성분으로 함유하는분자영상 진단제를 제공한다. In addition, a transgenic animal model transformed with the recombinant vector can be prepared. The transgenic animal model may produce a transgenic mouse using a mouse, and continuously monitor the expression level of the target cell by the secretory imaging reporter of the secreted target tracking fusion protein of the present invention expressed in the transgenic mouse. Can be. For example, constructing a transgenic mouse expressing a fusion protein of an RGD peptide and a secretion imaging reporter that tracks ανβ expressed in renal neovascularization enables continuous non-invasive imaging of neovascular sites. Screening for substances that inhibit development or tumor angiogenesis can be used as an animal model. When neovascularization occurs by implanting a tumor into the animal model, the secretion imaging reporter secreted from the cells of the animal model is localized at the tumor site. Imaging is possible, and therapeutic agents can be screened to reduce neovascularization of tumor tissues. According to one aspect of the present invention, the present invention provides a molecular imaging diagnostic agent containing the secreted target tracking fusion protein as an active ingredient.
본 발명의 분자영상 진단제에 있어서， 상기 분비형 표적 추적 융합 단백질은 서열번호 2의 아미노산 서열을 갖는 것을 특징으로 한다. 본 발명의 한 양태에 따르면， 본 발명은 상기 분비형 표적 추적 융합 단백질 을 유효성분으로 함유하는 종양 세포 치료제를 제공한다. In the molecular imaging diagnostic agent of the present invention, the secretory target tracking fusion protein is characterized by having an amino acid sequence of SEQ ID NO: 2. According to one aspect of the present invention, the present invention provides a tumor cell therapeutic agent containing the secreted target tracking fusion protein as an active ingredient.
상기 분비형 표적 추적 융합 단백질의 치료용 펩타이드 (예컨대， TRAIL)가 종양세포 치료에 유효함이 알려져 있으므로 [Argiris et al., Exp. Biol Med. Since the therapeutic peptide (eg, TRAIL) of the secreted target tracking fusion protein is known to be effective for treating tumor cells, Argiris et al., Exp. Biol Med.
(Maywood). May 1；236(5)：524-36(2011)], 상기 분비형 표적 추적 융합 단백질은 종 양 세포 치료제로 이용될 수 있다. 본 발명은 본 발명에 따른 분비형 표적 추적 융합 단백질을 유효성분으로 함 유하는 감염 또는 염증 질환 치료제를 제공한다. (Maywood). May 1; 236 (5): 524-36 (2011)], the secreted target tracking fusion protein can be used as a tumor cell therapy. The present invention provides an agent for treating an infectious or inflammatory disease containing a secreted target tracking fusion protein according to the present invention as an active ingredient.
상기 융합 단백질의 치료용 펩타이드 (예컨대， 디펜신 (defensins), 카텔리시딘 (cathelicidin) LL-37, MIP-3a/CCL20, 부포린 (buforin) I 또는 유비퀴시딘 (ubiquicidin))가 감염 또는 염증 질환 치료에 유효함이 알려져 있으므로 [Wiesner et al., Virulence. Sep-Oct; 1 (5) :440-64(2010)], 상기 분비형 표적 추적 융합 단백질은 감염 또는 염증 질환 치료제로 이용될 수 있다. 또한， 본 발명은 본 발명의 분비형 표적 추적 융합 단백질을 유효성분으로
함유하는 허혈 질환 치료제를 제공한다. Therapeutic peptides (eg defensins, cathelicidin LL-37, MIP-3a / CCL20, buforin I or ubiquicidin) of the fusion protein are infected or inflammatory. Since it is known to be effective in treating diseases, Wiesner et al., Virulence. Sep-Oct; 1 (5): 440-64 (2010)], the secreted target tracking fusion protein can be used as a therapeutic agent for infectious or inflammatory diseases. In addition, the present invention is the secreted target tracking fusion protein of the present invention as an active ingredient Provided is an ischemic disease treatment.
상기 융합 단백질의 치료용 펩타이드 (예컨대， VEGF( vascular endothelial growth factor), HGF (hepatocy te growth factor), 안지오포이에틴 (angiopoietin) 또는 PLGF(placental growth factor))가 허혈 질환 치료에 유효함이 알려져 있으므로 [Beohar et al, J. Am. Coll. Cardiol. Oct 12；56(16)：1287-97(2010)； Mac et al., Wiley. Interdiscip. Rev. Syst. Biol. Med. Nov— Dec;2(6):694-707(2010); Taniyama et al., Nippon Rinsho. May;68(5):949-52(2010)], 상기 분비형 표적 추적 융합 단백 질은 허혈 질환 치료제로 이용될 수 있다. 본 발명의 종양 세포 치료제， 감염 또는 염증 질환 치료제 또는 허혈 질환 치료제는 치료용 약제로 이용되기 위해서， 약제학적 분야에서 공지된 방법에 의해 제조될 수 있으며， 약학적으로 허용되는 담체， 부형제， 희석제 등과 혼합하여 분말， 과립， 정제， 캡술제， 또는 주사제 등의 제형으로 제조되어 사용될 수 있다. 또한 이들 들은 비 경구 투여 (예컨대， 정맥 내， 피하， 복강 내， 또는 국소에 적용)하거나 경구 투여될 수 있다. It is known that the therapeutic peptide of the fusion protein (eg, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), angiopoietin or PLGF (placental growth factor)) is effective in treating ischemic disease. As described in Beohar et al, J. Am. Coll. Cardiol. Oct 12; 56 (16): 1287-97 (2010); Mac et al., Wiley. Interdiscip. Rev. Syst. Biol. Med. Nov— Dec; 2 (6): 694-707 (2010); Taniyama et al., Nippon Rinsho. May; 68 (5): 949-52 (2010)], the secreted target trace fusion protein can be used as a therapeutic agent for ischemic disease. Tumor cell therapy, infection or inflammatory disease treatment or ischemic disease treatment agent of the present invention can be prepared by a method known in the pharmaceutical art for use as a therapeutic agent, a pharmaceutically acceptable carrier, excipient, diluent, etc. The mixture may be prepared and used in the form of powder, granules, tablets, capsulants, or injections. They may also be administered orally (eg, intravenously, subcutaneously, intraperitoneally, or topically) or orally.
본 발명의 상기 치료제들은 치료학적으로 유효한 양으로 투여한다. 상기 "치 료학적으로 유효한 양 (therapeutically effective amount)"은 의학적 치료에 적용 가 능한 합리적인 수혜 /위험 비율로 질환을 치료하기에 층분한 양을 의미하며， 환자의 연령， 성별, 체중， 건강상태， 질병의 증상, 투여시간， 투여방법에 따라 적절히 선택될 수 있으며， 바람직하게는 성인기준 1일 0.01 - 100 mg이 투여될 수 있다. The therapeutic agents of the invention are administered in a therapeutically effective amount. The term “therapeutically effective amount” means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and includes the age, sex, weight, health condition, It may be appropriately selected depending on the symptoms of the disease, the time of administration and the method of administration, preferably 0.01-100 mg per day of adult.
【유리한 효과】 Advantageous Effects
이상 설명한 바와 같이， 본 발명에 따르면 세포외 분비기능을 갖는 펩타이드， 분자영상 리포터 또는 치료용 펩타이드 또는 단백질 및 표적세포에 특이적 결합능
을 갖는 펩타이드로 이루어진 분비형 표적 추적 융합 단백질을 박테리아 또는 포유 류세포를 이용하여 제조할 수 있으며， 상기 융합 단백질은 표적세포에 특이적으로 결합할 수 있고， 결합 여부를 영상화하여 확인할 수 있다. 또한 유도촉진자를 이용 하여 상기 융합 단백질을 필요시에만 발현시킬 수 있다. 뿐만 아니라， 상기 융합 단 백질을 생산할 수 있는 면역세포주 둥을 구축함으로써 세포치료에도 적용이 가능하 다. 이외에도 본 발명은 치료용 펩타이드 또는 단백질을 이용하여 표적세포를 치료 할 수 있는 치료제를 검색할 수 있다. 따라서 본 발명의 분비형 표적 추적 융합 단 백질을 이용하면 분자영상 진단제 또는 종양 세포 치료제로서 유용하게 사용할 수 있으며， 치료제의 검색에 이용할 수 있을 것으로 사료된다. As described above, according to the present invention, specific binding ability to peptides, molecular imaging reporters or therapeutic peptides or proteins having extracellular secretory function and target cells Secreted target tracking fusion protein consisting of a peptide having a can be prepared using bacteria or mammalian cells, the fusion protein can specifically bind to the target cell, can be confirmed by imaging the binding. In addition, the induction promoter can be used to express the fusion protein only when necessary. In addition, it is possible to apply to cell therapy by building an immune cell cluster capable of producing the fusion protein. In addition, the present invention can search for a therapeutic agent that can treat a target cell using a therapeutic peptide or protein. Therefore, the secretory target tracking fusion protein of the present invention can be usefully used as a molecular imaging agent or a tumor cell therapeutic agent, and can be used to search for a therapeutic agent.
그리고 종양 이외의 심근경색 및 하지허혈을 포함한 허혈성질환， 망망증， 염 증질환 등 신생혈관 형성이 병의 발생 및 진행에 상관되는 질환의 분자영상 진단제 및 치료제， 및 치료제의 검색방법으로도 사용이 가능할 것으로 사료된다. It is also used as a diagnostic method for molecular imaging, treatment and treatment of neovascularization such as ischemic diseases, retinopathy, and inflammatory diseases including myocardial infarction and lower limb ischemia other than tumors. This is believed to be possible.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 본 발명의 분비형 표적 추적 융합 단백질의 모식도를 나타낸 것이다. 도 2는 pcDNA3.으 sGlu-mCherry-RGD><3 와 pcDNA3.0-sGluc-mCherry-cRGD 재조합 백터 구축 과정을 나타낸 모식도이다. 도 3은 상기 재조합 백터의 개열지도를 나타낸 것이다. Figure 1 shows a schematic of the secreted target trace fusion protein of the present invention. Figure 2 is a schematic diagram showing the construction of pcDNA3. SGlu-mCherry-RGD> <3 and pcDNA3.0-sGluc-mCherry-cRGD recombinant vector. Figure 3 shows a cleavage map of the recombinant vector.
도 4는 다양한 세포주에서 ανβ3 발현을 측정한 결과이다. 4 shows the results of measuring α ν β3 expression in various cell lines.
도 5는 본 발명의 융합 단백질을 박테리아와 배양시 배양배지 및 세포 용해 물에서의 단백질 발현을 측정한 결과이다. Figure 5 is the result of measuring the protein expression in culture medium and cell lysate when incubating the fusion protein of the present invention with bacteria.
도 6은 본 발명의 융합 단백질을 JM109 박테리아에 형질 전환시킨 후， 융합 단백을 박테리아 세포에서 분리 한 후， 단백질 발현을 측정한 결과 이다. 6 is a result of measuring the protein expression after transforming the fusion protein of the present invention to JM109 bacteria, after separating the fusion protein from bacterial cells.
도 7은 도 3의 재조합 백터를 CHO 세포주에 형질전환 시킨 후， mCheny 형
광으로 세포에 이입 여부를 관찰한 결과이다. 7 is transformed into the CHO cell line recombinant vector of Figure 3, mCheny type It is the result of observing the migration to the cell by light.
도 8은 본 발명의 융합 단백질을 포유동물세포 (CHO)와 배양시 배양배지 및 세포 용해물에서의 단백질 발현을 측정한 결과이다. Figure 8 is the result of measuring the protein expression in culture medium and cell lysate when incubating the fusion protein of the present invention with mammalian cells (CHO).
도 9은 발광효소 (Galusssia luciferase)를 이용하여 CHO 및 synovium 세포에 서 본 발명의 융합 단백질이 ανβ3에 특이적으로 결합하는지를 측정한 결과이다. 도 10은 sGluc— mCheny— RGDx3 융합단백질을 분비하는 포유류세포와 ανβ3 가 발현하는 synovium 세포를 함께 배양하여 공초점현미경 영상으로 형광 단백질 (mCherry fluorescent protein)을 이용하여 synovium 세포에만 본 발명의 융합 단 백질이 ανβ3에 특이적으로 결합하는 것을 증명한 결과이다. 9 is a result of measuring whether the fusion protein of the present invention specifically binds to ανβ3 in CHO and synovium cells using a luminescent enzyme (Galusssia luciferase). 10 is a fusion protein of the present invention only in synovium cells using a fluorescent protein (mCherry fluorescent protein) in a confocal microscope image by culturing together a mammalian cell secreting the sGluc—mCheny—RGDx3 fusion protein and a synovium cell expressing ανβ3. After a proof that specifically binding to the α ν β3 it is.
도 11는 sGluc— mCherry-RGD><3 융합단백질을 분비하는 포유류 세포와 ανβ 3가 발현하는 U87MG-EGFP 세포를 함께 배양하여 얻은 공초점현미경 영상으로， U87MG-EGFP 세포에 sGluc— mCheny-RGDx3가 부착되어 형광신호를 나타냄을 보 여주는 영상이다. FIG. 11 is a confocal microscopy image obtained by incubating sGluc—mCherry-RGD> <3 fusion protein and a U87MG-EGFP cell expressing ανβ 3 together with sGluc—mCheny-RGDx3 valent in U87MG-EGFP cells. It is an image showing the fluorescent signal attached.
도 12은 sGluc-mCherry 융합단백질을 분비하는 포유류 세포와 ανβ3가 발현 하는 U87MG—EGFP 세포를 함께 배양하여 얻은 공초점현미경 영상으로， ανβ3가 발 현되는 U87MG— EGFP 세포라도 표적 추적 기능을 가진 RGD가 없는 sGluc-mCherry 융합단백은 부착하지 못하여 형광신호를 나타내지 않음을 보여주는 영상이다. 12 is a confocal microscope image obtained by culturing a mammalian cell secreting the sGluc-mCherry fusion protein and U87MG—EGFP cells expressing α ν β3 together with U87MG—EGFP cells expressing α ν β3. The sGluc-mCherry fusion protein without RGD has no fluorescence signal due to failure to attach.
도 13는 sGluc-mCherry 융합단백질을 분비하는 포유류 세포에서 sGluc-mCherry-RGDx3 융합단백질을 분리 농축한 뒤 ανβ3가 발현되는 U87MG-EGFP 세포에 처리하여， 포유류 세포에서 분비되어진 융합 단백 sGluc-mCheny-RGDx3가 U87MG— EGFP 세포에 부착되어 형광신호를 나타냄을 보 여주는 영상이다. 13 is isolated and concentrated sGluc-mCherry-RGDx3 fusion protein from mammalian cells secreting sGluc-mCherry fusion protein, and then treated to U87MG-EGFP cells expressing α ν β3, fusion protein sGluc-mCheny secreted from mammalian cells -RGDx3 is attached to U87MG-EGFP cells and shows fluorescence signal.
도 14은 JM109 박테리아에서 생산된 sGluc-mCherry— RGDx3와
sGluc-mCheny를 분리 농축하여 αν 3가 발현되는 U87MG-EGFP 세포와 ανβ3가 발현되지 않는 CHO— EGFP 세포에 처리한 후 얻은 공초점 현미경영상이다. sGluc-mCherry-RGD 3 가 처리된 (a) 영상에서는 ανβ3가 발현되는 U87MG-EGFP 세포에서 특이적인 결합이 관찰되며, sGluc-mCheny 가 처리된 (b) 영상에서는 두세 포 모두에서 특이적 결합이 관찰되지 않는다. 이상의 결과는 ανβ3가 발현되는 U87MG-EGFP 세포에만 sGluc-mCheny-RGI 3 융합 단백질이 결합되는 것을 보여 주어， sGluc-mCheny-RGI 3융합단백이 ανβ3에 특이적으로 결합함을 보여준다. 도 15는 ανβ3가 발현되는 U87MG— EGFP 와 ανβ3가 발현되지 않는 CHO-EGFP 세포를 실험동물 마우스에 이식 하고, 2주 후 박테리아에서 분리한 본 발 명의 융합 단백질을 꼬리 정맥 주사 하여 융합 단백질이 ανβ3에 특이적으로 결합하는 지를측정한 결과이다. ανβ3가 발현되는 U87MG-EGFP에서만 영상신호가 검출된다. 14 shows sGluc-mCherry—RGDx3 produced from JM109 bacteria. sGluc-mCheny was isolated and concentrated to treat U87MG-EGFP cells expressing αν3 and CHO-EGFP cells not expressing ανβ3. In (a) images treated with sGluc-mCherry-RGD 3, specific binding was observed in U87MG-EGFP cells expressing ανβ3, and specific binding was observed in both cells in (b) images treated with sGluc-mCheny. It doesn't work. The above results show that the sGluc-mCheny-RGI 3 fusion protein binds only to U87MG-EGFP cells expressing ανβ3, indicating that the sGluc-mCheny-RGI 3 fusion protein specifically binds to α v β3. 15 is α ν β3 expression U87MG- EGFP and α ν β3 is not expressing CHO cell-EGFP fusion with the experimental animal implanted in mice, tail vein injection of the fusion protein isolated from bacteria, to name two weeks after that This is the result of measuring whether the protein specifically binds to α v β 3. An image signal is detected only in U87MG-EGFP in which αvβ3 is expressed.
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하， 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다ᅳ 이들 실시예 는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다. 심시예 1. 백터 (Vector) 제작 Hereinafter, the present invention will be described in more detail with reference to Examples. Since these Examples are only for illustrating the present invention, the scope of the present invention is not interpreted to be limited by these Examples. Examination Example 1. Creating a Vector
PCR을 이용하여 pcDNA3.1-sGluc vector (전남대학교 민정준 교수， Stanford University Dr. Paulmurgan)로부터 secretory gaussia lucif erase 유전자 부분을 증폭 시킨 후， 증폭된 상기 유전자를 pRSET(B) 백터 (Invitrogen)로 삽입하여 pRSET(B)-sGlu(secretory Glue) 백터를 제작하였다. 이후, RGEKArg-Gly—Asp)의 염기서열이 3번 반복 혹은 RGD가 세포내에서 cyclic 형태가 되어 지는 염기서열을 프라이머 결합반응 (annealing)을 하여 상보가닥을 이룬 RGD 염기서열 (RGD><3 혹은
cRGD)을 제조하였다. 또한， PCR을 이용하여 pmR— mCherry vector(Clonetech)에서 mCherry 유전자만을 증폭하고， 증폭된 상기 유전자를 pRSET(B) 백터에 삽입하여 pRSET(B) -mCherry 백터를 제작하였다. 이후, 제작된 pRSET(B)_mCherry 백터에 미리 제작해 놓은 RGD 염기서열 (RGDx3 혹은 cRGD)을 삽입하여 pRSET(B)ᅳ mCherry-RGDx3 혹은 pRSET(B)-mCheny— cRGD 백터를 제작하였다. 상기 새로 제작된 pRSET(B)-mCherry-RGD><3 혹은 pRSET(B)-mCheny— cRGD 백터에서 Kpn I 및 EcoRI 제한효소를 이용하여 mCherry— RG1 3 혹은 mCherry-cRGD 유 전자를 절단한 후， 절단된 유전자를 미리 제작해 놓은 pRSET(B)— sGlu 백터로 삽입 하여 pRSET(B)-sGlu— mCherry— RGDx3 혹은 pRSET(B)-sGluᅳ mCherryᅳ cRGD 백 터를 제작하였다. 상기 pRSET(B)— sGlu-mCherry-RGD><3 혹은 pRSET(B)-sGlu- mCherry-cRGD 백터를 BamH I 및 EcoRI 제한효소를 이용하여 sGlucᅳ mCheny- RGDx3 혹은 sGluc— mCheery-cRGD 유전자를 절단하여， pcDNA3.0 백터 (Invitrogen)로 삽입시키고， 최종산물인 pcDNA3.0-sGlu-mCherry-RGD><3 혹은 pcDNA3.0— sGlu一 mCherry- cRGD벡터를 구축하였다. After PCR amplifies the secretory gaussia lucif erase gene portion from pcDNA3.1-sGluc vector (Professor Min Jung-Jun, Stanford University Dr. Paulmurgan), and inserts the amplified gene into the pRSET (B) vector (Invitrogen). pRSET (B) -sGlu (secretory Glue) vectors were constructed. Thereafter, the base sequence of RGEKArg-Gly—Asp is repeated three times or the base sequence in which RGD becomes cyclic in the cell is primer-annealed to form a complementary RGD sequence (RGD><3 or cRGD) was prepared. In addition, PCR was used to amplify only the mCherry gene in the pmR—mCherry vector (Clonetech) and insert the amplified gene into the pRSET (B) vector to prepare a pRSET (B) -mCherry vector. Subsequently, pRSET (B) ᅳ mCherry-RGDx3 or pRSET (B) -mCheny—cRGD vector was prepared by inserting a previously prepared RGD sequence (RGDx3 or cRGD) into the prepared pRSET (B) _mCherry vector. After cleaving the mCherry-RG1 3 or mCherry-cRGD gene using Kpn I and EcoRI restriction enzymes in the newly prepared pRSET (B) -mCherry-RGD><3 or pRSET (B) -mCheny—cRGD vector, The cleaved gene was inserted into a previously prepared pRSET (B) —sGlu vector to prepare pRSET (B) -sGlu—mCherry—RGDx3 or pRSET (B) -sGlu ᅳ mCherry ᅳ cRGD vector. PRSET (B) —sGlu-mCherry-RGD><3 or pRSET (B) -sGlu-mCherry-cRGD vector was digested with BamH I and EcoRI restriction enzymes to sGluc ᅳ mCheny-RGDx3 or sGluc—mCheery-cRGD gene Then, it was inserted into the pcDNA3.0 vector (Invitrogen), and the final product, pcDNA3.0-sGlu-mCherry-RGD><3 or pcDNA3.0—sGlu 一 mCherry-cRGD vector was constructed.
상기 pcDNA3.으 sGhi-mCheny-RGDx3 혹은 pcDNA3.0— sGlu-mCheny- cRGD 백터 구축 과정은 도 2에 나타내었으며， 상기 재조합 백터의 개열지도는 도 3 에 나타내었다. 실시예 2. 다양한 세포주에서의 ανβ3 발현 The pcDNA3. SGhi-mCheny-RGDx3 or pcDNA3.0—sGlu-mCheny-cRGD vector construction process is shown in Figure 2, the cleavage map of the recombinant vector is shown in Figure 3. Example 2. ανβ3 Expression in Various Cell Lines
여러 세포주에서 ανβ3의 발현을 확인하기 위해， 유방암 세포주 (MDA— MB— 231)， 폐암 세포주 (A549), 사람활막세포주 (2046 및 2047) 및 CHO (Chinese hamster ovary cell)로부터 RNA를 분리하였다. 먼저， 각각의 세포를 100 mm dish에 배양 후， Trizol(Invitrogen)을 이용하여 세포에서 RNA* 추출한 후， 분 리한 각각의 RNA를 RT-PCR 기법을 이용하여 cDNA를 합성한 후， αν 및 β3의 프
라이머 (primer; 특정 서열에 대하여 상보적인 짧은 가닥)를 이용하여 PCR과 DNA 전기영동을 통하여 mRNA 발현을 확인하였다. RNA was isolated from breast cancer cell lines (MDA—MB—231), lung cancer cell lines (A549), human synovial cell lines (2046 and 2047), and Chinese hamster ovary cells (CHO) to confirm the expression of ανβ3 in various cell lines. First, each cell was cultured in a 100 mm dish, RNA * was extracted from the cells using Trizol (Invitrogen), and then each RNA was isolated and synthesized cDNA using RT-PCR. F MRNA expression was confirmed by PCR and DNA electrophoresis using primers (short strands complementary to specific sequences).
그 결과， 도 4에 나타낸 바와 같이 RT-PCR을 통한 mRNA 발현은 CHO 세 포를 제외한 모든 세포에서 αν 및 β3의 발현이 전기영동 상으로 확인되었으나, CHO는 αν 및 β3 모두 전기영동 상에서 확인한 결과 발현되지 않았다. 실시예 3. 융합 단백질 발현 분석 As a result, as shown in FIG. 4, the expression of mRNA through RT-PCR was confirmed by the electrophoresis of αν and β3 in all cells except CHO cells, but the expression of CHO was confirmed by electrophoresis on both αν and β3. It wasn't. Example 3. Fusion Protein Expression Assay
pRSET(B) 백터는 JM109 박테리아에서 단백질 발현이 되므로， 이를 이용하면 표적세포의 추적이 가능한 다량의 분비형 표적 추적 융합 단백질을 얻을 수 있다. Since the pRSET (B) vector is expressed in JM109 bacteria, it can be used to obtain a large amount of secreted target tracking fusion protein that can be traced to target cells.
pcDNA 백터는 포유동물세포에서 단백질 발현이 되므로， 이를 이용하여 표적 세포 추적이 가능한 다량의 분비형 표적 추적 융합 단백질을 얻을 수 있으며， 세포 기반 치료시 이용 가능한 백터이기도 하다. Since the pcDNA vector is expressed in mammalian cells, it can be used to obtain a large amount of secreted target tracking fusion protein capable of tracking target cells, and is also a vector that can be used for cell-based treatment.
3 1. 박테리아 배양 시 3 1.Bacteria culture
본 발명의 분비형 표적 추적 융합 단백질을 생산할 수 있는 박테리아를 제작 하여 함께 배양시킨 후 배양액으로의 방출 여부를 확인하였다. 먼저， 실시예 1에서 제작한 pcDNA3.0— sGlu— mCheny-RGD><3 백터를 competent cell(DH5a)에 형질전환 시킨 후， 50 yg/ml의 암피실린 (ampicillin)을 넣고 2 ml만을 취하여 37 °C 쉐이킹 인 큐베이터에서 밤새도록 배양하였다. 배양 완료 후， 2 ml 중 100 μΐ만을 취하여 1500 rpm에서 5 분간 원심분리한 후, 상둥액을 새로운 류브에 "옮겨 담았다. 상등액을 제 거하고 남은 침전된 세포 펠렛 (cell pellet)에 세포 용해 버퍼 (5x lysis buffer, promega) 100 μΐ를 넣고 볼텍스 (voltex) 하였다. 이후， 1500 rpm에서 2 분간 다시 원 심분리한 후， 상등액 증 20 μΐ를 96-웰 루미노미터 플레이트 (96- well luminometer plate)에 넣었다. 이후 상기 플레이트에 gaussia luciferase substrate 100 μΐ를 넣고
루미노미터 (luminometer)로 확인하였다. Bacteria that can produce the secreted target trace fusion protein of the present invention were prepared and incubated together, and then released into the culture medium. First, the pcDNA3.0—sGlu—mCheny-RGD><3 vector prepared in Example 1 was transformed into competent cells (DH5a), 50 yg / ml of ampicillin was added, and only 2 ml was taken at 37 °. Incubated overnight in C shaking incubator. After completion of the culture, 2 after only 100 μΐ ml of centrifuged for 5 minutes at 1500 rpm ml, captured transferred to the dungaek the new ryubeu "supernatant to remove remaining after the precipitated cell pellet (cell pellet) in cell lysis buffer ( 100 μl of 5x lysis buffer, promega was added and vortexed, then centrifuged again at 1500 rpm for 2 minutes, and 20 μΐ of supernatant was added to a 96-well luminometer plate. Then add 100 μΐ of gaussia luciferase substrate to the plate. It was confirmed by a luminometer.
측정 결과， 도 5에 나타낸 바와 같이 세포배양 배지와 세포내의 루시퍼라제 (luciferase)의 활성도는 세포내의 루시퍼라제 활성도 보다 세포배양 배지에서의 루 시퍼라제 활성도가 8배 이상 높았다. As shown in FIG. 5, the activity of the cell culture medium and the intracellular luciferase was 8 times higher in the luciferase activity in the cell culture medium than the intracellular luciferase activity.
그리고 pRSET(B)-sGluc-mCheny一 RGDx3 또는 pRSET(B)— sGluc— mCherry-cRGD 백터를 JM109 박테리아에 이입하면 상기 융합 단백질을 배양액 및 박테리아에서 동일한 방법으로 얻을 수 있다. The fusion protein can be obtained in the same manner in culture and bacteria by introducing pRSET (B) -sGluc-mCheny RGDx3 or pRSET (B) -sGluc-mCherry-cRGD vector into JM109 bacteria.
JM109 박테리아에 pRSET(B)-sGluc-mCheny— RGDX3 또는 pRSET(B)- sGluc-mCheny-cRGD를 각각 이입하여 대량의 융합 단백질을 생성하고 His— tag 방 법을 이용한 정제 방법으로 상기 융합 단백만을 정제 할 수 있다. pRSET(B)— sGluc— mCherry— RGDX3 백터가 이입된 JM109 박테리아에서 발현된 sGluc-mCherry-RGDX3 융합단백과 pRSET(B)-sGluc-mCheny 백터가 이입된 JM109 박테리아에서 발현된 sGluc— mCherry 융합단백을 분리 정제하여 96—웰 플레 이트에 각 0.5ug 과 lug을 넣고 gaussia luciferase substrate 100 μΐ 넣고 루시퍼라 제의 활성도를 IVIS 영상으로 확인 하였다. PRSET (B) -sGluc-mCheny—RGDX3 or pRSET (B) -sGluc-mCheny-cRGD were introduced into JM109 bacteria, respectively, to generate a large amount of fusion proteins, and the fusion protein was purified by a purification method using His—tag method. can do. sGluc-mCherry-RGDX3 fusion protein expressed in pRSET (B)-sGluc- mCherry- RGDX3 vector grafted sGluc-mCherry-RGDX3 fusion protein and pRSET (B) -sGluc-mCheny vector sGluc- mCherry fusion protein expressed After separation and purification, 0.5ug and lug were added to the 96-well plate, 100 μl of gaussia luciferase substrate was added, and luciferase activity was confirmed by IVIS image.
측정 결과 도 6에서 나타낸 바와 같이 상기 융합 단백의 농도가 증가할수록 루시퍼라제의 활성이 증가 하는 것을 확인 할 수 있다. As shown in Figure 6 it can be seen that as the concentration of the fusion protein increases the activity of luciferase increases.
3-2.포유동물세포 배양 시 3-2.Incubating mammalian cells
본 발명의 분비형 표적 추적 융합 단백질을 생산할 수 있는 포유동물세포를 제작하여 함께 배양시킨 후 배양액으로의 방출 여부를 확인하였다. 먼저， CHO— sGlucᅳ mCheny-RGD를 발현하는 안정 세포주 (stable cell line)를 구축하기 위 해， ανβ3의 발현이 없는 CHO 세포에 실시예 1에서 제작된 pcDNA3O-sGlu一 mCherry-RGDx3 백터를 리포좀 (Lipofectamine, Invitrogen)을 이용하여 세포에 이입
한 후, 계대 배양하여 유세포 분석기로 mCheny 형광을 이용하여 세포내로 PCDNA3.0— sGlu-mCheny-RGI 3가 안전하게 이입된 세포만을 분류하였다 (도 7). 이후 상기 제작된 CHO-sGluc-mCherry— RGD가 안정적으로 이입된 CHO 세포주와 모세포인 대조군 CHO 세포를 24—웰 플레이트에 5xl04/well이 되도록 세포를 접종 하였다. 세포 접종 24 시간 후, 상기 웰의 배지 (media)를 새로운 튜브에 옮겨 담고， 플레이트 내에있는 세포는 PBS로 2번 세척하였다. 이후 세포 용해 버퍼 (5χ lysis buffer, promega) 100 ul를 넣어 플레이트 바닥에 있는 세포를 용해시켰다. 용해시킨 세포를 새로운 E-tube에 담고 1500 rpm에서 2 분간 원심분리하였다. 상등액을 96一 웰 루미노미터 플레이트에 각각 20 μΐ씩 넣고, 상기에서 튜브에 옮겨 담은 배지 (media)를 상기 플레이트에 20 μΐ씩 더 첨가하였다. 이후 gaussia luciferase substrate 100 μΐ 넣고 루미노미터 Guminometer)로 확인하였다. Mammalian cells were produced that could produce the secreted target tracking fusion protein of the present invention and cultured together to confirm their release into the culture. First, in order to construct a stable cell line expressing CHO—sGluc ᅳ mCheny-RGD, the pcDNA3O-sGlu 一 mCherry-RGDx3 vector produced in Example 1 was applied to CHO cells without ανβ3 expression. Incorporation into cells using Lipofectamine, Invitrogen) Subsequently, subcultures were used to classify only cells into which PCDNA3.0—sGlu-mCheny-RGI 3 was safely introduced into cells using mCheny fluorescence with a flow cytometer (FIG. 7). Then, the prepared CHO-sGluc-mCherry—RGC was stably inoculated with CHO cell line stably immobilized with CHO cell line and parental control CHO cells in a 24-well plate at 5xl0 4 / well. 24 hours after cell inoculation, the media of the wells were transferred to new tubes and the cells in the plates were washed twice with PBS. Then, 100 ul of cell lysis buffer (5 χ lysis buffer, promega) was added to lyse the cells at the bottom of the plate. Lysed cells were placed in fresh E-tubes and centrifuged for 2 minutes at 1500 rpm. 20 μl each of the supernatant was put into 96-well well luminometer plates, and 20 μl each of the media transferred to the tube was added to the plate. Then 100 μΐ gaussia luciferase substrate was added and confirmed by a luminometer Guminometer.
측정 결과, 도 8에 나타낸 바와 같이 세포배양 배지와 세포내의 루시퍼라제 (luciferase)의 활성도는 어떤 유전자도 이입하지 않은 모세포 CHO 세포에 비해 CHO— sGluc-mChenyᅳ RGD가 안정적으로 이입된 CHO 세포의 루시퍼라제 활성도는 30 배， 세포배양 배지에서의 루시퍼라제 활성도는 500 배 이상 높았다. 실시예 4. 욥합 단백질의 ανβ3 특이 결합 확인 As a result, as shown in Fig. 8, the cell culture medium and intracellular luciferase activity of lucifer of CHO-sGluc-mCheny® RGD stably immobilized compared to parental CHO cells without any genes The lyase activity was 30 times higher and the luciferase activity in cell culture medium was 500 times higher. Example 4. Confirmation of ανβ3 Specific Binding of the Job Protein
4-1. 발광효소 (Glusssia luciferase) 이용 4-1. Use of Lusssia luciferase
6-웰 플레이트에 CHO 및 synovium 세포를 5xl04/well로 접종한 후， 다음날 CHO-sGluc-mCheny-RGD를 배양 증에 얻은 배양 배지를 상기 두 종류의 세포가 각각 부착된 플레이트에 2 ml/well씩 넣었다. 48 시간 동안 배양시킨 후， PBS로 2번 세척하였다. 이후， PBS를 각 웰에 1 ml/well로 넣어주고， coelentrazine(0.25 ug in 50 μΐ PBS)를 넣어준 후， IVIS 영상을 획득 (도 9의 (A))하였다. 이때, 발광효소를 이 용한 이유는, IVIS 영상장비로 mCherry 형광 단백질의 신호를 영상화 하기 어려우
며, 발광효소가 형광 단백질보다 민감도가 높아 발광효소를 이용하였다. 살아 있는 동물에서 영상신호를 통해 평가하기 위해서는 민감도가 높은 영상신호가 필요하나， mCherry 보다 발광신호의 영상신호 민감도가 더 높다. After inoculating CHO and synovium cells with 5xl0 4 / well in a 6-well plate, the culture medium obtained in the culture of CHO-sGluc-mCheny-RGD the next day was 2 ml / well on a plate to which the two types of cells were attached. Put in. After incubation for 48 hours, the cells were washed twice with PBS. Thereafter, PBS was added to each well at 1 ml / well, coelentrazine (0.25 ug in 50 μΐ PBS) was added thereto, and an IVIS image was obtained (FIG. 9A). In this case, the reason for using luminescent enzyme is that it is difficult to image the signal of mCherry fluorescent protein with IVIS imaging equipment. In addition, luminase was more sensitive than fluorescent protein and luminase was used. In order to evaluate a video signal in a live animal, a high sensitivity video signal is required, but the sensitivity of the light signal is higher than that of mCherry.
측정 결과， 도 8에 나탄낸 바와 같이 ανβ3가 발현되는 synovial 세포 (2052)는 ανβ3를 발현되지 않는 CHO 세포에 비해 20 배 가량의 발광영상신호가 증가되는 결과를 보였다 (도 9의 (Β)). 따라서 상기 융합 단백질이 ανβ3에 특이적으로 결합되 었음을 알 수 있다. Measurements, synovial cells 2052 that ανβ3 is expressed as embellish Nathan in Figure 8 showed the results that increasing the luminescence image signal of about two times compared to CHO not expressing α ν β3 cells (Fig. 9 (Β )). Therefore, it can be seen that the fusion protein is specifically bound to α ν β3.
4-2. 형광 단백질 (mCheny fluorescent protein) 이용 4-2. Using mCheny fluorescent protein
Nunc(Lab Tek-Chamber slide)에 CHO, CHO— sGluc-mCheny— RGD, synovium 세포 (2052 세포)를 각각 접종한 후, CHO + synovium 군， CH으 RGD + synovium 군을 각각 1><104 세포로 혼합하여 각 웰에 접종하였다. 48 시간 동안 배 양시킨 후， 따뜻한 PBS로 2번 세척하였다. 이후， BD cytofix/cytoperm 1χ buffer 100 μΐ를 상기 각 웰에 넣고， 4 °C에서 30분 동안 고정시켰다. 그리고 15 ml 튜브에 BD perm/wash buffer 1 ml과 PBS 9 ml을 넣어 1χ Perm/wash 버퍼를 제조하였 다. 상기 제조한 lx Perm/wash 버퍼 200 μΐ를 웰당 넣어 2번 세척하였다. Anti-human av^3(milipore, 0.5 ug/100 yl in 1 BD Perm/wash 버퍼)를 1 시간 실 온에 두었다가， 상기 각 웰당 lx BD Perm/wash 버퍼 (실온에 두었던 antibody를 washing하는 버퍼) 200 μΐ를 넣고 3번 세척하였다. Secondary antibody FITC Goat-anti mouse(BD, 2 ug/100 μΐ in l BD Perm/wash 버퍼)를 40 분간 실은에 두었다가， 상기 각 웰당 1χ BD Perm/wash 버퍼 (secondary antibody를 washing하 는 버퍼) 200 μΐ를 넣고 3번 세척하였다. 이후, DAPI를 웰당 한방울씩 떨어뜨려 염 색시킨 후, 커버 글라스를 덮어 mounting 하였다. 염색된 세포를 공초점 현미경 (Confocal microscopy)으로 관찰하였다.
측정 결과， 도 10에 나타낸 바와 같이， 세포의 핵을 DAPI 염색을 통하여 관 찰할 수 있었으며， 현미경의 붉은색 파장으로 세포내 안전하게 이입된 mCheny 형 광단백질을 확인하고， 초록색 필터를 통해 ανβ3가 발현하는 세포임을 확인할 수 있 었다. 그것 중 분비형 단백질이 2052 세포에 결합하여 발현하는 세포를 볼 수 있었 으며， 이를 이미지 결합을 통해 확인할 수 있었다. 즉， ανβ3가 발현되는 세포에 mCherry 형광이 관찰되어， 상기 분비형 표적 추적 융합 단백질이 ανβ3가 발현되는 세포에 결합되었음을 알 수 있었다. 실시예 5. 욥합 단백질의 ανβ3 추적 기능 확^ CHO, CHO—sGluc-mCheny—RGD and synovium cells (2052 cells) were inoculated on Nunc (Lab Tek-Chamber slide), and then 1><10 4 cells were added to CHO + synovium group and CH RGD + synovium group, respectively. Each well was inoculated by mixing. After 48 hours of incubation, the cells were washed twice with warm PBS. Then, 100 μΐ of BD cytofix / cytoperm 1 χ buffer was added to each well and fixed at 4 ° C. for 30 minutes. Then, 1 χ Perm / wash buffer was added to 1 ml of BD perm / wash buffer and 9 ml of PBS in a 15 ml tube. 200 μl of the prepared lx Perm / wash buffer was added twice per well and washed twice. Anti-human av ^ 3 (milipore, 0.5 ug / 100 yl in 1 BD Perm / wash buffer) was kept at room temperature for 1 hour, and then lx BD Perm / wash buffer (buffer for washing antibody at room temperature) 200 for each well. μΐ was added and washed three times. Secondary antibody FITC Goat-anti mouse (BD, 2 ug / 100 μΐ in l BD Perm / wash buffer) was placed in the package for 40 minutes, then 1 χ BD Perm / wash buffer (buffer for washing the secondary antibody) for each well 200 μΐ was added and washed three times. After that, DAPI was stained by dropping one drop per well, and then the cover glass was covered and mounted. Stained cells were observed by confocal microscopy. As a result of the measurement, as shown in FIG. 10, the nucleus of the cells could be observed through DAPI staining, and the mCheny-type photoproteins were safely introduced into the cells at the red wavelength of the microscope, and ανβ3 was expressed through the green filter. It was confirmed that the cells. Among them, secretory protein was found to bind to and express 2052 cells, which could be confirmed through image binding. That is, mCherry fluorescence was observed in the cells expressing ανβ3, indicating that the secretory target tracking fusion protein was bound to the cells expressing ανβ3. Example 5 Confirmation of ανβ3 Tracking Function of Job-Jobs
5-1. CHO-sGluc-mCherry-RGDx3 융합 단백질의 ανβ3 추적 5-1. Ανβ3 Tracking of the CHO-sGluc-mCherry-RGDx3 Fusion Protein
ανβ3가 발현되는 U87MG 세포에 EGFP가 발현되는 세포주를 구축시킨 enhanced GFP를 세포질내에 발현하는 U87MG 세포 (U87MG-EGFP)와 본 발명의 융합 단백질 CHO— sGluc-mCherry-RGDx3 세포 (sGluc— mCherry— RGD><3 분비하는 CHO 세포)를 각각 5><104， ΙχΙΟ5의 개수로 혼합하여 IWAKI Glass based dish에 접 종하여 48 시간 동안 혼합배양 한 후， 공초점 현미경 (Confocal microscopy)으로 관 찰하였다. U87MG cells (U87MG-EGFP) expressing enhanced GFP in the cytoplasm of U87MG cells expressing ανβ3 expressed in the cytoplasm and the fusion protein CHO—sGluc-mCherry-RGDx3 cells (sGluc—mCherry—RGD) of the present invention. <3 secreting CHO cells) were mixed in a number of 5><10 4 , Ι χ ΙΟ 5 , inoculated in an IWAKI Glass-based dish for 48 hours, and then observed by confocal microscopy. It was.
현미경 측정 결과는 도 11에 나타내았다. 좌상 영상 (A)은 GFP 파장으로 관 찰한 결과로서 U87MG-EGFP 세포가 관찰되며， 우상 영상 (B)은 mCherry를 관찰하 기 위해 Red 파장으로 관찰한 결과로서 CHO-sGluc— mCheny— RGDx3 세포가 관찰 됨을 확인할 수 있으며， GFP 파장에 U87MG-EGFP 세포가 관찰되던 시야 증간부 위에 붉은색 영상신호가 관찰된다 (우하 영상 (D)). 이는 CHO— sGluc-mCheny- RGDx3 세포에서 분비된 sGluc-mCheny— RGDx3 분비형 표적 추적 융합 단백질이 ανβ3가 발현되는 U87MG-EGFP세포를 추적함을 증명하는 결과이다.
5-2. CHO-sGluc— mCherry 융합 단백질의 ανβ3 추적 The microscopic measurement results are shown in FIG. 11. The upper left image (A) was observed with GFP wavelength and U87MG-EGFP cells were observed. The upper right image (B) was observed with red wavelength to observe mCherry and CHO-sGluc— mCheny— RGDx3 cells. In addition, a red image signal is observed on the visual field center where U87MG-EGFP cells were observed at the GFP wavelength (bottom image (D)). This demonstrates that the sGluc-mCheny—RGDx3 secreted target tracking fusion protein secreted from CHO—sGluc-mCheny-RGDx3 cells tracks U87MG-EGFP cells expressing ανβ3. 5-2. Ανβ3 Tracking of CHO-sGluc—mCherry Fusion Proteins
ανβ3가 발현되는 U87MG 세포에 EGFP가 발현되는 세포주를 구축시킨 enhanced GFP를 세포질내에 발현하는 U87MG 세포 (U87MG— EGFP)와 융합 단백질 CHO-sGluc-mCherry 세포 (sGluc— mCherry 분비하는 CHO 세포)를 각각 5><104， ΙχΙΟ5의 개수로 혼합하여 IWAKI Glass based dish에 접종하여 48 시간 동안 혼합 배양 한 후， 공초점 현미경 (Confocal microscopy)으로 관찰하였다. U87MG cells (U87MG—EGFP) expressing enhanced GFP in the cytoplasm of U87MG cells expressing ανβ3 expressed in the cytoplasm (U87MG—EGFP) and fusion protein CHO-sGluc-mCherry cells (sGluc—mHO cells secreting CHO cells), respectively. ><10 4 ， Ι χ ΙΟ 5 were mixed and inoculated in an IWAKI Glass based dish, mixed incubation for 48 hours, and observed by confocal microscopy (Confocal microscopy).
현미경 측정 결과는 도 12에 나타내었다. 좌상 영상 (A)은 GFP 파장으로 관 찰한 결과로서 U87MG-EGFP 세포가 관찰되며， 우상 영상 (B)은 mCherry를 관찰하 기 위해 Red 과장으로 관찰한 결과로 CH으 sGlucᅳ mCherry 세포가 관찰됨을 확인 할 수 있다. 하지만， GFP 파장에 세포가 관찰되던 시야 중간부위에 붉은색의 영상신호가 관찰되지 않은 것으로 보아 (도 10과 비교)， 분비형 융합 단백 질에 ανβ3 표적 추적 기능을 가진 RGD 부분을 제거하면 ανβ3 발현 세포를 추적하 지 못하는 것을 알 수 있다. The microscopic measurement results are shown in FIG. 12. The upper left image (A) was observed with GFP wavelength, and U87MG-EGFP cells were observed, and the upper right image (B) was observed with red exaggeration to observe mCherry. can do. However, the red image signal was not observed in the middle of the field of view where cells were observed at the GFP wavelength (compare FIG. 10). It can be seen that the cells cannot be tracked.
이러한 결과는， CHO-sGluc-mCheny-RGD><3 세포에서 분비된 sGluc-mCherry-RGDx3 분비형 표적 추적 융합 단백질 증 RGD 부분이 ανβ3의 표 적 추적기능의 특이성을 보여주는 결과이며， 또한 본 발명의 융합 단백질인 sGluc-mCheny-RGD<3의 신생혈관추적 특이성을 보여준다, These results indicate that the RGD portion of the sGluc-mCherry-RGDx3 secreted target tracking fusion protein gene secreted from CHO-sGluc-mCheny-RGD> <3 cells shows the specificity of the target tracking function of ανβ3. Shows angiogenic specificity of sGluc-mCheny-RGD <3, a fusion protein
5-3. CHO-sGluc-mCherry-RGD 3 융합 단백질의 ανβ3 추적 5-3. Ανβ3 Tracking of CHO-sGluc-mCherry-RGD 3 Fusion Protein
ανβ3가 발현되는 U87MG 세포에 EGFP가 발현되는 세포주를 구축시킨 enhanced GFP를 세포질내에 발현하는 U87MG 세포 (U87MG—EGFP)를 5><104의 개 수로 IWAKI Glass based dish에 접종하고, CH으 sGluc-mCherry— RGDx3 세포만을 배양하였던 dish에서 배양액을 채취하여 상기 U87MG 세포를 접종한 dish에 넣고 배양한 후, 공초점 현미경으로 관찰하였다.
현미경 측정 결과는 도 13에 나타내었다. 좌상 영상 (A)은 GFP 파장으로 관 찰한 결과로서 U87MG— EGFP 세포가 관찰되며， 우상 영상 (B)은 mCherry를 관찰하 기 위해 Red 파장으로 관찰한 결과로서 GFP 파장에 U87MG— EGFP 세포가 관찰되 던 동일한 시야 증간 부위에 붉은색 영상신호가 관찰됨을 확인할 수 있다. 이는 sGluc-mCherry-RGD 3 분비형 표적 추적 융합 단백질이 ανβ3을 발현하는 세포를 추적하는 기능이 있음을 보여준다. U87MG cells (U87MG—EGFP) expressing enhanced GFP in the cytoplasm of U87MG cells expressing ανβ3 expressed in the cytoplasm were inoculated in an IWAKI Glass based dish in a number of 5><10 4 , and sGluc- The culture solution was taken from a dish in which only mCherry—RGDx3 cells were cultured and placed in a dish inoculated with the U87MG cells, followed by culturing, followed by confocal microscopy. The microscopic measurement results are shown in FIG. 13. The upper left image (A) shows the U87MG—EGFP cells as a result of observation at the GFP wavelength, and the upper right image (B) shows the U87MG—EGFP cells at the GFP wavelength as the result of observing the mCherry. In the same field of vision, a red image signal is observed. This shows that the sGluc-mCherry-RGD 3 secreted target tracking fusion protein has the ability to track cells expressing ανβ3.
5-4. 박테리아에서 생산된 융합 단백질의 ανβ3 추적 5-4. Ανβ3 Tracking of Fusion Proteins Produced in Bacteria
ανβ3가 발현되는 U87MG 세포와 ανβ3가 발현되지 않는 CHO세포를 실험동 물 마우스에 5X106 의 개수로 이식 한 후， 이식 2주후 종양이 생성된 후 박테리아에 서 생산된 융합 단백질을 꼬리 정맥을 통하여 20ug 주사한다. 주사 1시간 후, 루시 퍼라제 기질을 꼬리 정맥으로 주사하여 IVIS 영상을 획득 하였다. ανβ3가 발현되는 U87MG 세포에서만 영상신호를 획득할 수 있었고 이는 sGluc-mCheny— RGDx3 분 비형 표적 추적 융합 단백질이 생체내에서도 ανβ3을 발현하는 세포를 추적하는 기 능이 있음을 보여준다. U87MG cells expressing ανβ3 and CHO cells not expressing ανβ3 were transplanted into 5 × 10 6 cells in experimental animals, and after 2 weeks of implantation, 20ug injection of the fusion protein produced by bacteria through the tail vein. do. One hour after injection, IVIS images were obtained by injecting luciferase substrate into the tail vein. to obtain an image signal only in U87MG cells which express the ανβ3 were This shows that the capability to keep track of cells expressing α ν β3 sGluc-mCheny- RGDx3 min-free target tracking fusion protein within the living body.
【산업상 이용가능성】 Industrial Applicability
이상 설명한 바와 같이， 본 발명에 따르면 세포외 분비기능을 갖는 펩타이드， 분자영상 리포터 또는 치료용 펩타이드 또는 단백질 및 표적세포에 특이적 결합능 을 갖는 펩타이드로 이루어진 분비형 표적 추적 융합 단백질을 박테리아 또는 포유 류세포를 이용하여 제조할 수 있으며， 상기 융합 단백질은 표적세포에 특이적으로 결합할 수 있고, 결합 여부를 영상화하여 확인할 수 있다. 또한 유도촉진자를 이용 하여 상기 융합 단백질을 필요시에만 발현시킬 수 있다. 뿐만 아니라， 상기 융합 단 백질을 생산할 수 있는 면역세포주 등을 구축함으로써 세포치료에도 적용이 가능하
다. 이외에도 본 발명은 치료용 펩타이드 또는 단백질을 이용하여 표적세포를 치료 할 수 있는 치료제를 검색할 수 있다. 따라서 본 발명의 분비형 표적 추적 융합 단 백질을 이용하면 분자영상 진단제 또는 종양 세포 치료제로서 유용하게 사용할 수 있으며， 치료제의 검색에 이용할 수 있을 것으로 사료된다. As described above, according to the present invention is a bacterial or mammalian cell comprising a secretory target tracking fusion protein consisting of an extracellular secretory function, a molecular image reporter or therapeutic peptide or protein, and a peptide having a specific binding ability to a target cell. It can be prepared using, the fusion protein can specifically bind to the target cell, can be confirmed by imaging the binding. In addition, the induction promoter can be used to express the fusion protein only when necessary. In addition, it is possible to apply to cell therapy by building an immune cell line capable of producing the fusion protein. All. In addition, the present invention can search for a therapeutic agent that can treat a target cell using a therapeutic peptide or protein. Therefore, the secretory target tracking fusion protein of the present invention can be usefully used as a molecular imaging agent or a tumor cell therapeutic agent, and can be used to search for a therapeutic agent.
그리고 종양 이외의 심근경색 및 하지허혈을 포함한 허혈성질환， 망망증， 염 증질환 둥 신생혈관 형성이 병의 발생 및 진행에 상관되는 질환의 분자영상 진단제 및 치료제， 및 치료제의 검색방법으로도 사용이 가능할 것으로 사료된다.
Ischemic diseases, including myocardial infarction and lower limb ischemia, and nephropathy, including inflammatory diseases, can be used as molecular imaging diagnostics and treatments for diseases related to the development and progression of the disease. This is believed to be possible.
Claims
【청구항 1】 [Claim 1]
(a) 세포외 분비기능을 갖는 펩타이드， (b) 분자영상 리포터 또는 치료용 펩 타이드 또는 단백질， 및 (c) 표적세포에 특이적 결합능을 갖는 펩타이드로 이루어진 분비형 표적 추적 융합 단백질. A secretory target tracking fusion protein comprising (a) a peptide with extracellular secretory function, (b) a molecular image reporter or therapeutic peptide or protein, and (c) a peptide with specific binding ability to a target cell.
【청구항 2】 [Claim 2]
제 1항에 있어서, 상기 세포외 분비기능을 갖는 펩타이드는 분비형 가우시아 루시퍼ᅳᅵ라제 (secretory gaussia luciferase; Sec-Glue), 분비형 항체 (secretory antibody), 또는 신호 인식 입자 (signal recognition particle)에 의해 인식되는 소수성 의 아미노산 서열을 포함하는 펩타이드로 구성된 군에서 선택된 펩타이드인 것을 특징으로 하는 분비형 표적 추적 융합 단백질. The method of claim 1 wherein the peptide having the extracellular secretion is secreted Gaussian cyano Lucifer eu cyclase (secretory gaussia luciferase; Sec-Glue ), secreted antibodies (secretory antibody), or a signal recognition particle (signal recognition particle) Secreted target tracking fusion protein, characterized in that the peptide selected from the group consisting of a peptide comprising a hydrophobic amino acid sequence recognized by.
【청구항 3] [Claim 3]
제 2항에 있어서， 상기 세포외 분비기능을 갖는 펩타이드는 서열번호 3의 아 미노산 서열을 갖는 분비형 가우시아 루시퍼라제 (Sec-Glue)인 것을 특징으로 하는 분비형 표적 추적 융합 단백질. The secretory target tracking fusion protein according to claim 2, wherein the extracellular secretory peptide is secreted Gaussian luciferase (Sec-Glue) having the amino acid sequence of SEQ ID NO: 3.
【청구항 4】 [Claim 4]
제 1항에 있어서， 상기 분자영상 리포터 또는 치료용 펩타이드 또는 단백질 은 형광 단백질 (fluorescent protein), 발광 단백질 (bioluminescent protein), 광음영상 단백질 (photoacoustic protein), 방사성핵종 및 방사선조영제， 초음파조영제 또는 MR 조영제가 결합하는 단백질로 구성된 군에서 선택된 분자영상 리포터 또는 TRAIL, apoptin, 사이토라이신 (cytolysin), INF-β, FASL, TNF-α, 사이토카인 (IL—2 또는
IL-18), 혈관신생억제 펩타이드 (anti— angiogenic peptide - thrombospondin, endostatin), 허혈치료 펩타이드 (pro— angiogenic peptide- vascular endothelial growth factor, hepatocyte growth factor, angiopoietin, placental growth factor) 또 는 디펜신 (defensin) 및 카텔리시딘 (cathelicidin)과 같은 항균성 (antimicrobial) 펩타 이드들로 구성된 군에서 선택된 치료용 펩타이드 또는 단백질인 것을 특징으로 하 는 분비형 표적 추적 융합 단백질. The method of claim 1, wherein the molecular image reporter or therapeutic peptide or protein is a fluorescent protein, a bioluminescent protein, a photoacoustic protein, radionuclides and radiographic agents, ultrasound contrast agents or MR contrast agents. Molecular image reporter or TRAIL, apoptin, cytolysin, INF-β, FASL, TNF-α, cytokine (IL—2 or) selected from the group consisting of IL-18), anti-angiogenic peptide (thrombospondin, endostatin), ischemic peptide (pro— angiogenic peptide- vascular endothelial growth factor, hepatocyte growth factor, angiopoietin, placental growth factor) or defensin (defensin) And a therapeutic peptide or protein selected from the group consisting of antimicrobial peptides such as cathelicidin).
【청구항 5】 [Claim 5]
제 4항에 있어서， 상기 분자영상 리포터는 서열번호 4의 아미노산 서열을 갖 는 mCherry이며, 상기 치료용 펩타이드 또는 단백질은 서열번호 6의 아미노산 서열 을 갖는 TRAIL인 것을 특징으로 하는 분비형 표적 추적 융합 단백질. According to claim 4, wherein the molecular image reporter is mCherry having an amino acid sequence of SEQ ID NO: 4, the therapeutic peptide or protein is secreted target tracking fusion protein, characterized in that the TRAIL having an amino acid sequence of SEQ ID NO: 6 .
【청구항 6】 [Claim 6]
제 1항에 있어서， 상기 분자영상 리포터 또는 치료용 펩타이드는 분자영상 리포터 또는 치료용 물질과 결합능을 갖는 펩타이드인 것을 특징으로 하는 분비형 표적 추적 융합 단백질. According to claim 1, wherein the molecular imaging reporter or therapeutic peptide secretion-type target tracking fusion protein, characterized in that the peptide having a binding capacity with the molecular imaging reporter or therapeutic material.
【청구항 7】 [Claim 7]
계 1항에 있어서， 상기 표적세포에 특이적 결합능을 갖는 펩타이드는 RGD, NGR (종양혈관신생 targeting), Apopep- 1 (apoptosis targeting peptide), GPC-3(glypican-3) 표적 펩타이드 (간암， 생식선암， 혹색종 등에서 과발현되는 GPC-3 추적 펩타이드), IL-12 표적 펩타이드， 아미노펩티다아제 (aminopeptidase) P 표적 펩타이드， IL11— γ 표적 펩타이드， 뉴클레오린 (Nucleolin) 표적 펩타이드 또는 종양 림프절 (tumor lymphatic) 표적 펩타이드로 구성된 군에서 선택된 펩타이드인
것을 특징으로 하는 분비형 표적 추적 융합 단백질. The method of claim 1, wherein the peptide having a specific binding capacity to the target cells are RGD, NGR (tumor angiogenesis targeting), Apopep-1 (apoptosis targeting peptide), GPC-3 (glypican-3) target peptide (liver cancer, reproduction GPC-3 tracer peptide overexpressed in adenocarcinoma, melanoma, etc.), IL-12 target peptide, aminopeptidase P target peptide, IL11—γ target peptide, Nucleolin target peptide or tumor lymphatic A peptide selected from the group consisting of target peptides A secretory target tracking fusion protein, characterized in that.
【청구항 8】 [Claim 8]
제 7항에 있어서, 상기 표적세포에 특이적 결합능을 갖는 펩타이드는 서열번 호 5의 아미노산 서열을 갖는 RGD 또는 서열번호 7의 아미노산 서열을 갖는 cRGD 인 것을 특징으로 하는 분비형 표적 추적 융합 단백질. The secretory target tracking fusion protein according to claim 7, wherein the peptide having a specific binding ability to the target cell is RGD having an amino acid sequence of SEQ ID NO: 5 or cRGD having an amino acid sequence of SEQ ID NO: 7.
【청구항 9】 [Claim 9]
제 1항에 있어서, 상기 세포외 분비기능을 갖는 펩타이드는 서열번호 3의 아 미노산 서열, 분자영상 리포터 또는 치료용 펩타이드 또는 단백질은 서열번호 4 또 는 서열번호 6의 아미노산 서열， 및 표적세포에 특이적 결합능을 갖는 펩타이드는 서열번호 5 또는 서열번호 7의 아미노산 서열을 갖는 것을 특징으로 하는 분비형 표적 추적 융합 단백질. According to claim 1, wherein the peptide having an extracellular secretion function is the amino acid sequence of SEQ ID NO: 3, molecular imaging reporter or therapeutic peptide or protein is specific for the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, and target cells A peptide having a potent binding capacity is a secreted target tracking fusion protein, characterized in that having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.
【청구항 10] [Claim 10]
제 9항에 있어서, 상기 분비형 표적 추적 융합 단백질은 서열번호 2 또는 서 열번호 9의 아미노산 서열을 갖는 ανβ3 표적 추적 융합 단백질인 것을 특징으로 하 는 분비형 표적 추적 융합 단백질. 10. The secretory target tracking fusion protein of claim 9, wherein the secretory target tracking fusion protein is an ανβ3 target tracking fusion protein having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 9.
【청구항 11】 [Claim 11]
제 9항에 있어서， 상기 분비형 표적 추적 융합 단백질은 MMP-2(Matrix Metalloproteinase-2) 또는 MMP-9에 의해 절단될 수 있는 펩타이드 서열을 더 포 함하는 것을 특징으로 하는 분비형 표적 추적 융합 단백질.
10. The method of claim 9, wherein said secreted target tracking fusion protein further comprises a peptide sequence that can be cleaved by Matrix Metalloproteinase-2 (MMP-2) or MMP-9 secreted target tracking fusion protein .
【청구항 12] [Claim 12]
제 1항 내지 제 11항 증 어느 한 항에 따른 분비형 표적 추적 융합 단백질을 코딩하는 유전자. A gene encoding a secreted target tracking fusion protein according to any one of claims 1 to 11.
【청구항 13】 [Claim 13]
제 12항에 있어서， 상기 유전자는 서열번호 1 또는 서열번호 8의 염기서열을 갖는 특징으로 하는 유전자. The gene of claim 12, wherein the gene has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 8.
【청구항 14】 [Claim 14]
제 12항에 있어서, 상기 유전자는 유도형 촉진자 (inducible promoter)를 더 포 함하는 것을 특징으로 하는 유전자. 13. The gene of claim 12, wherein the gene further comprises an inducible promoter.
【청구항 15】 [Claim 15]
제 12항 내지 제 14항에 따른 유전자를 포함하는 재조합 백터. Recombinant vector comprising the gene according to claim 12.
【청구항 16】 [Claim 16]
제 15항에 있어서， 상기 재조합 백터는 도 3의 개열지도를 갖는 pRSET(B)— sGluc— mCherry— RG] 3, pRSET(B)_sGluc— mCherry-cRGD, pcDNA3.0-sGluc-mCherry-RGDx3, 또는 pcDNA3O-sGluc— mCherry-cRGD 백터인 것을 특징으로 하는 재조합 백터. The method of claim 15, wherein the recombinant vector is pRSET (B)-sGluc-mCherry-RG] 3, pRSET (B) _sGluc-mCherry-cRGD, pcDNA3.0-sGluc-mCherry-RGDx3 having a cleavage map of FIG. Or pcDNA3O-sGluc—mCherry-cRGD vector.
【청구항 17】 [Claim 17]
제 15항에 따른 재조합 백터로 형질전환된 형질전환 세포주.
A transformed cell line transformed with the recombinant vector according to claim 15.
【청구항 18] [Claim 18]
제 17항에 있어서， 상기 형질전환된 세포주는 CHO-sGluc-mCheny-RGD 또 는 CHO— sGluc— mCherry-cRGD인 것을 특징으로 하는 형질전환 세포주. 18. The transformed cell line of claim 17, wherein said transformed cell line is CHO-sGluc-mCheny-RGD or CHO-sGluc-mCherry-cRGD.
【청구항 19] [Claim 19]
제 15항에 따른 재조합 백터로 형질전환된 형질전환 동물모델, A transgenic animal model transformed with a recombinant vector according to claim 15,
【청구항 20] [Claim 20]
제 19항에 있어서， 상기 형질전환된 동물모델은 형질전환 마우스인 것을 특 징으로 하는 형질전환 동물모델. The transgenic animal model of claim 19, wherein the transgenic animal model is a transgenic mouse.
【청구항 21】 [Claim 21]
제 1항에 따른 분비형 표적 추적 융합 단백질을 유효성분으로 함유하는 분자 영상 진단제. A molecular imaging diagnostic agent comprising the secreted target tracking fusion protein according to claim 1 as an active ingredient.
【청구항' 22】 [Claim port '22]
제 21항에 있어서， 상기 분비형 표적 추적 융합 단백질은 서열번호 2의 아미 노산 서열을 갖는 것을 특징으로 하는 분자영상 진단제. The diagnostic agent for molecular imaging according to claim 21, wherein the secretory target tracking fusion protein has an amino acid sequence of SEQ ID NO: 2.
【청구항 23】 [Claim 23]
제 1항에 따른 분비형 표적 추적 융합 단백질을 유효성분으로 함유하는 종양 세포 치료제.
A tumor cell therapeutic agent comprising the secreted target tracking fusion protein according to claim 1 as an active ingredient.
【청구항 24】 [Claim 24]
제 1항에 따른 분비형 표적 추적 융합 단백질을 유효성분으로 함유하는 감염 또는 염증 질환의 진단 및 치료제. An agent for the diagnosis and treatment of an infectious or inflammatory disease comprising the secretory target tracking fusion protein according to claim 1 as an active ingredient.
【청구항 25] [Claim 25]
제 1항에 따른 분비형 표적 추적 융합 단백질을 유효성분으로 함유하는 허혈 질환의 진단 및 치료제.
An agent for diagnosing and treating ischemic disease, comprising the secreted target tracking fusion protein according to claim 1 as an active ingredient.
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| KR20100127798A (en) * | 2008-02-27 | 2010-12-06 | 예다 리서치 앤드 디벨럽먼트 캄파니 리미티드 | Rgd(bacterio)chlorophyll conjugates for photodynamic therapy and imaging of necrotic tumors |
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