WO2012165927A2 - Protéine de fusion sécrétoire pour suivi de cible - Google Patents

Protéine de fusion sécrétoire pour suivi de cible Download PDF

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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
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peptide
fusion protein
target tracking
target
seq
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PCT/KR2012/004401
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Korean (ko)
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WO2012165927A3 (fr
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안병철
이재태
이상우
황미혜
김상엽
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경북대학교 산학협력단
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Publication of WO2012165927A3 publication Critical patent/WO2012165927A3/fr

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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; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified 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
    • 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
    • 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
    • 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
    • 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
    • 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.

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Abstract

La présente invention concerne une protéine de fusion sécrétoire pour le suivi d'une cible, et plus spécifiquement une protéine de fusion sécrétoire pour le suivi d'une cible comprenant les éléments suivants : un peptide présentant une sécrétion extracellulaire; un rapporteur d'imagerie moléculaire ou une protéine ou un peptide thérapeutique; et un peptide présentant une capacité de liaison spécifique à une cellule cible. Selon la présente invention, étant donné que la protéine de fusion sécrétoire pour le suivi d'une cible peut être préparée pour comprendre un peptide avec une sécrétion extracellulaire, un rapporteur d'imagerie moléculaire ou une protéine ou un peptide thérapeutique, et un peptide possédant une capacité de liaison spécifique à une cellule cible, ladite protéine de fusion peut se lier spécifiquement à une cellule cible, et l'occurrence de liaison peut être vérifiée par imagerie. En outre, l'expression de la protéine de fusion peut être induite au moyen d'un promoteur d'induction, uniquement lorsque cela est nécessaire. Par ailleurs, il est possible de rechercher un agent thérapeutique apte à traiter la cellule cible au moyen de ladite protéine ou dudit peptide thérapeutique. C'est pourquoi la protéine de fusion sécrétoire pour le suivi de cible de la présente invention est considérée comme utile en tant qu'agent de diagnostic d'imagerie moléculaire ou agent thérapeutique de cellule tumorale.
PCT/KR2012/004401 2011-06-02 2012-06-04 Protéine de fusion sécrétoire pour suivi de cible WO2012165927A2 (fr)

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KR101705353B1 (ko) * 2013-10-17 2017-02-13 경북대학교 산학협력단 ApoPep-1 펩타이드 프로브를 포함하는 골관절염 조기진단용 시약

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KR20100127798A (ko) * 2008-02-27 2010-12-06 예다 리서치 앤드 디벨럽먼트 캄파니 리미티드 괴사종양의 광역학요법 및 영상용 알지디(박테리오)클로로필 컨쥬게이트

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017081082A3 (fr) * 2015-11-09 2017-08-24 Curevac Ag Molécules d'acide nucléique optimisées

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