KR101471475B1 - Fused peptide type tumor inhibitor, tumor diagnosing agent and method for preparing the same - Google Patents

Abstract

A fusion type peptide type tumor suppressor excellent in cell membrane permeability, a tumor diagnostic agent and a method for producing the same. The tumor suppressor has an amino acid sequence of SEQ ID NO: 3 (cRGDyK-S-S-CPLHSpT). Herein, the tumor suppressor is a prodrug in which a PLHSpT peptide (Pro-Leu-His-Ser-p-Thr, SEQ ID NO: 1) and a cRGDyK peptide (cyclic Arg-Gly-Asp-D-Tyr- Linked fused peptide. In addition, the present invention provides a tumor diagnostic agent comprising a peptide having the amino acid sequence of SEQ ID NO: 3 and a detectable label (label) bonded thereto.

Description

Fused Peptide Tumor Inhibitors, Tumor Diagnostic Agents, and Methods of Producing the same

The present invention relates to a fusion peptide, and more particularly, to a fusion peptide type tumor suppressor excellent in cell membrane permeability, a tumor diagnostic agent and a method for producing the same.

Polo-like kinase 1 (Plk1) is a regulator of cell cycle progression in the mitosis process of cells, and is a C-terminal polo-box domain (polo-like kinase 1) -box domain (PBD), a binding motif that regulates kinase activity. Plk is overexpressed in many types of cancer cells and is considered an antimitotic target of cancer cells. Ser-p-Thr phosphopeptide (PLHSpT phosphopeptide, SEQ ID NO: 1, wherein p-Thr is a phosphorylated (< Phosphorylated, PO ₃ H ₂ ^- ) Thr) is known, and the PLHSpT peptide stops mitosis in cancer cells and induces apoptotic cell death. That is, the PLHSpT peptide strongly binds to the PBD domain, which is an activity regulatory region of Plk-1 kinase specifically expressed in cancer cells, and inhibits the cell division of cancer cells (US Patent Publication No. 20110104713; Nat Struct Mol Biol. 2011 Apr , 18 (4): 516; Nat Struct Mol Biol 2009, 16: 876-82). As described above, the PLHSpT peptide has excellent anticancer effect but can not penetrate the membrane of cancer cells (such as HeLa cells) (that is, it can not penetrate into cells). Therefore, in order to obtain an anticancer effect, Direct micro-injection is required. Therefore, PLHSpT peptide itself has a disadvantage that it is difficult to be used as a practical cancer drug.

It is an object of the present invention to provide a fusion peptide type tumor suppressor capable of overcoming the disadvantages of the conventional PLHSpT peptide having low cell membrane permeability and having excellent cell membrane permeability and effectively inhibiting the proliferation of cancer cells.

It is another object of the present invention to provide a tumor diagnostic agent capable of acquiring a tumor image by specifically binding to cancer cells.

It is still another object of the present invention to provide a method for producing the tumor suppressor and the tumor diagnostic agent.

In order to achieve the above object, the present invention provides a tumor suppressor having the amino acid sequence of SEQ ID NO: 3 below.

[SEQ ID NO: 3]

cRGDyK-S-S-CPLHSpT

Herein, the tumor suppressor is a peptide comprising a PLHSpT peptide (Pro-Leu-His-Ser-p-Thr, SEQ ID No. 1) and a cRGDyK peptide (cyclic Arg-Gly-Asp-D-Tyr- Linked fused peptide.

In addition, the present invention provides a tumor diagnostic agent comprising a peptide having the amino acid sequence of SEQ ID NO: 3 and a detectable label (label) bonded thereto. The present invention also relates to a method for producing SPDP-activated cRGDyK by binding N-succinimidyl-3- (2-pyridyldithio) -propionate (SPDP) to the lysine side chain of cRGDyK peptide step; And binding the SPDP-activated cRGDyK to the N-terminal cysteine of the CPLHSpT peptide. The present invention also provides a method for producing a fusion peptide having the amino acid sequence of SEQ ID NO: 3.

The tumor suppressor according to the present invention can effectively inhibit the proliferation of cancer cells in vivo and in vitro and is useful as a radiation tracer for the diagnosis of radiolabeled tumors.

Brief Description of the Drawings Fig. 1 is a schematic diagram for explaining a process in which the fusion peptide type tumor suppressor according to the present invention exhibits an anti-cancer effect in the body. Fig.
FIG. 2 is a view showing a process of synthesizing a fusion peptide according to an embodiment of the present invention. FIG.
FIG. 3A is a graph showing the cell viability of human cervical cancer cells treated with cRGDyK, PLHSpT, cRGDyK-Asp-CPLHSpT, and cRGDyK-SS-CPLHSPT.
FIG. 3B is a photograph (magnification: 200) showing the morphology of cells after treatment of human hybridoma cells and human cervical cancer cells with cRGDyK, PLHSpT, and cRGDyK-SS-CPLHSPT.
FIG. 3c shows the results of PLHSpT and cRGDyK-SS-CPLHSpT uptake of human cervical cancer cells by adding PLHSpT and cRGDyK-SS-CPLHSpT treated with fluorescent material (FITC) and staining the nuclei of human cervical cancer cells with DAPI With a fluorescence microscope.
FIG. 4A is a graph showing the results of measuring the inhibitory effect of Plk-1 kinase on PLSpT and cRGDyK-S-S-CPLHSpT after treatment of human squamous cell and human cervical cancer cells.
FIG. 4B is a graph showing changes in Plk-1 kinase inhibitory effect over time after treatment with human HSCs and human cervical cancer cells with PLHSpT and cRGDyK-S-CPLHSpT.
FIG. 4c is a fluorescence microscope photograph showing the centrosome and spindle states after treatment of human hybridoma cells and human cervical cancer cells with PLHSpT and cRGDyK-S-CPLHSpT.
FIG. 5A is a photograph of Western blot analysis of PARP separation after treating human hybridoma cells and human cervical cancer cells with cRGDyK, PLHSpT, and cRGDyK-SS-CPLHSpT.
FIG. 5 is a graph showing the results of FACS analysis of cell death after treating human cervical cancer cells with FITC-cRGDyK, FITC-PLHSpT, and FITC-cRGDyK-SS-CPLHSpT.
6A is a graph showing tumor size changes after treatment of tumor mice with PLHSpT and cRGDyK-SS-CPLHSpT.
Figure 6b shows tumor photographs after 19 days after treatment of tumor mice with PLHSpT and cRGDyK-SS-CPLHSpT.
FIG. 6c is a photograph showing the results of tumor cells treated with PLHSpT and cRGDyK-SS-CPLHSpT, followed by staining tumor tissue after 19 days.
FIG. 7 is a photograph showing tumor cell death by coloring tumor tissues with Annexin V and propidium iodide (PI) after treating tumor mice with PLHSpT and cRGDyK-SS-CPLHSpT.
FIG. 8A is a graph showing the results of evaluating cell binding force over time after treatment with human cervical cancer cells and human cervical cancer cells with radiation labeled cRGDyK-SS-CPLHSpT.
8B is a photograph showing the results of PET scanning over time after treatment of tumor mice with radiolabeled cRGDyK-SS-CPLHSpT.
Figure 8c is a graph showing the in vivo distribution of the radioactive tracer after treatment of the tumor mouse with a radioactive tracer (radiolabeled cRGDyK-SS-CPLHSpT).

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The fusion peptide type tumor suppressor according to the present invention is an improvement of a conventional cancer drug targeting polo-box domain (PBD) of pollo-type kinase 1 (Plk1). Specifically, the tumor suppressor according to the present invention has the amino acid sequence of SEQ ID NO: 3 below and is characterized in that the PLHSpT peptide (Pro-Leu-His-Ser-p-Thr, SEQ ID NO: And a selectively targeting cRGDyK peptide (cyclic Arg-Gly-Asp-D-Tyr-Lys, SEQ ID NO: 2) are linked via cysteine (C) via a disulfide bond.

[SEQ ID NO: 3]

cRGDyK-S-S-CPLHSpT

The tumor suppressor of SEQ ID NO: 3 enables integrin receptor-specific targeting and cell internalization to the PLHSpT peptide, which inhibits the activity of Plk-1 kinase and exhibits an anti-cancer effect, thereby selectively targeting cancer cells The PLHSpT derivative having the cGGDyK peptide fused thereto has excellent anticancer effect and is easily internalized or inserted into the cell by the fused RGD moiety. The tumor suppressor according to the present invention can be used not only for the peptide of the amino acid sequence shown in SEQ ID NO: 3 but also for a part of the amino acid sequence shown in SEQ ID NO: 3 as long as it has anti-cancer effect and intracellular transitivity, Deleted or modified variants thereof.

Arginine-glycine-aspartate (RGD) peptides are α _v β ₃ integrin receptor inhibitors that increase expression levels in cancer cells (Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 1994, 264: 569-71; Definition of two angiogenic pathways by distinct alpha v integrins. Science 1995, 270: 1500-2). The [alpha] _{v [} beta] ₃ integrin is a cell adhesion protein generally located in the vasculature of solid tumors and is expressed in the angiogenesis process of the tumor. Thus, the? _V ? ₃ integrin is one of the biomarkers useful for targeting tumors (J Immunol 2006, 176: 6225-34) . The peptide containing the RGD sequence (such as an? _V ? ₃ integrin inhibitor) has been used as a tumor diagnostic contrast agent through tumor imaging due to tumor specificity and selectivity, has a high? _V ? ₃ integrin binding capacity, (Nucl Med Biol 2004, 31: 179-89; Bioconjug Chem 2004, 15: 41-9; Cancer Res 2002, 62 : 6146-51). Bioconjugation based on disulfide bonds (-SS-) is a method of binding agents used in drug delivery systems. The disulfide bonds used in the peptide fusion of the present invention are reversibly bound to and separated from each other to promote drug delivery to the cytosol through the cell membrane to have a tumor specific cleavable property. Therefore, in the fusion peptide of the present invention, a disulfide covalent bond is used so that it is attached to a target point of the cell, migrates into the cell, and then separates and releases the drug into the cell interior space.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a hypothetical schematic diagram for explaining a process in which the fusion peptide type tumor suppressor according to the present invention exhibits an anti-cancer effect in the body. FIG. As shown in FIG. 1, the fused peptide (cRGDyK-SS-CPLHSpT) is mediated through the? _V ? ₃ integrin receptor, passed through the cell membrane, The disulfide bonds are cleaved (enzymatic cleavage) to produce free CPLHSpT. The free CPLHSpT peptide is transferred and bound to the PBD domain, which is the nuclear binding site of the nucleus, and inhibits cell mitosis, thus exhibiting an anticancer effect. Looking at this in more detail, when cRGDyK- fusion polypeptide of the invention is supplied in a particular tumor, in combination with the α _v β ₃ integrin receptor (integrin receptor) moves into the cell. Within the cell, it is believed that the disulfide bond of the conjugated peptide is separated and the CPLHSpT phosphopeptide and the cRGDyK peptide are separated. The isolated CPLHSpT phosphopeptide binds to PBD of plk1, effectively inhibiting cancer cell proliferation. On the other hand, when the PLHSpT peptide having insufficient cell membrane permeability or the fusion peptide not separating was used, the effect of inhibiting cancer cell proliferation was not shown (see Experimental Example 1). Therefore, in order for the PLHSpT peptide component to inhibit PBD, a cleavable linker capable of inducing the PLHSpT peptide component into the cell is required.

The fusion peptide (PLHSpT derivative) according to the present invention inhibits the activity of Plk-1 kinase and the cell division of cancer cells by attaching to the PBD domain of pololine kinase 1 (Plk1) whose expression is increased in cancer cells, It exhibits anticancer effect by the apoptosis mechanism of tissue. In the fusion peptide of the present invention, the intracellular transfer (transfer) of PLHSpT is spontaneous, and the diagnosis and treatment of cancer cells are effectively carried out by PLHSpT transfected into cells, and thus the selectivity of tumor target is excellent. In addition, the fusion peptide of the present invention is useful as a cancer treatment agent (anticancer agent) because of its low toxicity in vivo, and exhibits excellent diagnostic and anti-cancer effects particularly in human glioma subtype and cervical cancer. The fusion peptide according to the present invention is a targeted drug delivery system that permeates a receptor membrane mediated cell membrane.

Again, referring to FIG. 1, the isolated residues (cRGDyK and CPLHSpT) are confined within cancer cells, so that the location of the cancer cells can be detected if the separated residues are radiolabeled. Therefore, when a detectable label (label) is bound (attached or denatured) to the fusion peptide according to the present invention, cancer cells present in the living body can be directly imaged to effectively diagnose cancer (tumor). That is, the fusion peptide according to the present invention can be used as a tumor diagnostic agent (tumor target radioactive tracker) as it binds to a detectable label. Examples of the detectable label include radioactive isotopes (for example, ⁶⁸ Ga, ¹⁸ F, ¹²³ I, ¹²⁴ I, and ⁶⁴ Cu ¹¹¹ In), fluorescent light emitters, and the like. Also, if desired, the detectable label may be labeled with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane- , N "'- tetraacetic acid), as described above, to the fusion peptide. For example, the fusion peptide according to the present invention may be labeled with a radioactive isotope, injected into an animal tail vein into which the tumor has been implanted, and then irradiated with radioactive (radioactive) radioactive isotopes using an appropriate detection device such as positron emission tomography When the isotope is detected, an image (tumor image) of cancer cells distributed in the body can be obtained from the PET image. In addition to the radioactive isotope, the fusion peptide according to the present invention can be combined with various detectable detectable labels (fluorescent labels), and besides PET, another detection device capable of detecting bound labels An optical image can be obtained. The fusion peptide according to the present invention is excellent in the diagnostic effect of the tumor even when used at a low concentration. Since the fusion peptide according to the present invention targets PBD of Plk1, it is useful for diagnosis and confirmation of tumor in vivo or in vitro by labeling with ⁶⁸ Ga or the like.

The fusion peptide according to the present invention can be produced by various conventional synthetic methods. 2 is a view illustrating a process of synthesizing a fusion peptide according to an embodiment of the present invention. As shown in FIG. 2, in order to synthesize the fusion peptide (cRGDyK-SS-CPLHSPT) according to the present invention, the amino acid necessary for the solid-phase resin is sequentially added, Prepare the peptide. On the other hand, N-succinimidyl-3- (2-pyridyl dithio) -propionate (SPDP) was added to the lysine side chain of cRGDyK peptide Conjugated to prepare SPDP-activated cRGDyK. The SPDP-activated cRGDyK is bound to the N-terminal cysteine of the CPLHSpT peptide to synthesize cRGDyK-SS-CPLHSpT. Thus, SPDP is used as a heterobifunctional linker to link the lysine residue of cRGDyK to the cysteine portion of CPLHSpT via a disulfide bond. By combining these two peptides having different functions by the solid-phase peptide reaction, it is possible to avoid various disadvantages associated with the organic synthesis of the peptide.

A detectable marker (label) can be attached to the thus synthesized cRGDyK-SS-CPLHSpT fusion peptide by a conventional method to produce a radioactive tracker, that is, a tumor diagnostic agent. For example, the cRGDyK-SS-CPLHSpT fusion peptide and the SCN-Bz-NOTA (2- (p-isothiocyanatobenzyl) -1,4,7-triazacyclononane-1,4,7-triaceticacid, triActaclononane-1,4,7-triacetic acid) to synthesize NOTA-cRGDyK-SS-CPLHSpT, and labeling the NOTA-bound peptide with a label such as ⁶⁸ Ga to prepare a radioactive tracer for tumor diagnosis can do.

The fusion peptide according to the present invention may be used as a composition together with one or more pharmaceutically acceptable carriers. Examples of pharmaceutically acceptable carriers include saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and mixtures thereof. If necessary, antioxidants, buffers, And the like. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. And further formulated in a suitable manner in the art. The fusion peptide according to the present invention or a composition comprising it may be administered intravenously, intraperitoneally, intramuscularly, subcutaneous, intradermal, nasal, mucosal ), Inhalation, and oral route, as described above. The dosage varies depending on the subject's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of disease. For example, the daily dosage is about 0.01 to 500 mg / Kg, preferably 0.1 to 100 mg / kg, more preferably 0.5 to 50 mg / kg, and can be administered once a day or divided into several times a day.

The present invention provides a method for treating or suppressing a tumor in which a peptide having an amino acid sequence of SEQ ID NO: 3 (including its variants) is administered as an active ingredient to a human or animal. The present invention also relates to a method for diagnosing a tumor which comprises administering to a human or animal a tumor diagnostic agent comprising a peptide having an amino acid sequence of SEQ ID NO: 3 (including its variants) and a detectable label to provide.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited by the following Examples.

[Example 1] Synthesis of cRGDyK-SS-CPLHSPT peptide

(1) Amino acids to be synthesized in Rink amide resin were put in order in accordance with a standard Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase protocol and reacted in DMF (dimethyl formamide) solution to obtain CPLHSpT Peptide was synthesized (see Fig. 2). 4 equivalents of HOBt (hydroxybenzotriazole), HBTU (O-benzotriazole-N, N, N '-tetramethyluronium-hexafluoro-phosphate) and DIEA (diisopropylethylamine) were added as a coupling reagent. Respectively. After the reaction, the resin and the peptide are cleaved by treatment with a solution of trifluoroacetic acid (TFA), and the desired peptide is separated and purified from the obtained peptides by high performance liquid chromatography (HPLC) Respectively. Mass / assisted laser desorption / ionization time-of-flight mass spectrometry was used to determine the mass of the separated and purified peptide. As a result, m / z of the CPLHSpT peptide was 736.3. Similarly, the PLHSpT peptide was prepared and the m / z = 632.62 of the PLHSpT peptide produced.

(2) To the lysine side chain of the protected -cRGDyK (62.5 mg, 63.5 mmoL, FutureChem, Korea) in basic solvent (DMSO) solvent and basic condition (DIEA) SPDP-activated cRGDyK was prepared by conjugating N-succinimidyl-3- (2-pyridyl dithio) -propionate (SPDP; 25 mg, 80.0 mmol) . The mass of the separated and purified SPDP-activated cRGDyK was determined by MALDI-TOF mass spectrometry and found to be m / z = 1181.60. In the DMF solvent, the SPDP-activated cRGDyK (37 mg, 31.3 mmol) was coupled to the N-terminal cysteine of Fmoc-CPLHSpT (77 mg, 50.0 mmol) to synthesize cRGDyK-SS-CPLHSpT. The mass of the separated and purified cRGDyK-SS-CPLHSpT was confirmed by MALDI-TOF mass spectrometry and was found to be m / z = 1440.57.

[Example 2] Synthesis of ⁶⁸ Ga-labeled radioactive tracer

Synthesis of cRGDyK-SS-CPLHSpT (14 mg, 9.72 mmol) and SCN-Bz-NOTA (2- (p-isothiocyanatobenzyl) -1,4,7-triazacyclononane-N, N "-triacetic acid trihydrochloride, = 1,4,7-triazacyclononane-N, N ', N "-triacetic acid, 6 mg, 10.72 mmol) was reacted in 0.1 M sodium carbonate buffer (pH 9.5) to synthesize NOTA-cRGDyK-SS-CPLHSpT , And purified and separated by HPLC. The mass of the separated and purified peptide was confirmed by MALDI-TOF mass spectrometry and was found to be m / z = 1890.08. The novel conjugated or activated peptide was labeled with ⁶⁸ Ga to produce a novel radioactive tracer for tumor diagnosis. Specifically, ⁶⁸ Ga was eluted from a ⁶⁸ Ge / ⁶⁸ Ga generator system and then the obtained ⁶⁸ Ga solution (74 MBq, 0.8 mL of 0.1 N HCl solution) was added to the NOTA-bound peptide (NOTA- cRGDyK-SS-CPLHSpT). 0.5 M phosphate buffer (0.3 mL) was added to adjust the pH of the reaction solution to 6.0 to 6.5, followed by reaction at room temperature for 10 to 20 minutes. Then, the reaction mixture was subjected to size exclusion chromatography To obtain a radioligand compound ⁶⁸ Ga-NOTA-cRGDyK-SS-CPLHSpT labeled with radioactive isotope ⁶⁸ Ga. The yield of the decay-corrected radioactive tracer ⁶⁸ Ga-NOTA-cRGDyK-SS-CPLHSpT was approximately 20-35%. ^{Since 68} Ga is obtained from the ⁶⁸ Ge / ⁶⁸ Ga generator, a radioisotope used for PET scanning can be economically produced without using an expensive cyclotron.

[Experimental Example 1] Evaluation of the effect of cRGDyK-SS-CPLHSpT (XTT assay)

Human cervical cancer cells (HeLa cells) were cultured in 96-well plates for 24 hours at a cell number of 1 x 10 ³ per well. At this time, DMEM (Dulbecco's Modified Eagle Medium) medium, 10% FBS (Fetal Bovine Serum) and 1X penicillin / streptomycin solution were used as the culture medium. The cultured cells were washed with the culture medium without FBS and then cRGDyK, PLHSpT, cRGDyK-Asp-CPLHSpT and cRGDyK-SS-CPLHSPT were added to each well at a concentration of 1 μM, 10 μM, 20 μM, 50 μM And 100 [mu] M, and reacted for 24 hours. Next, 10% XTT solution (Sigma-Aldrich, USA) was added as a cell proliferation reagent and further reacted for 4 hours.

After the reaction, the colorimetric intensity of each well was measured at a wavelength of 475 nm, and cell viability was evaluated. The results are shown in FIG. 3A. As shown in FIG. 3A, when cRGDyK-SS-CPLHSPT ( 1 in FIG. 3A) was added, tumor cell survival rate was significantly higher than that in the case where other peptides (cRGDyK, PLHSpT, cRGDyK-Asp-CPLHSpT) Respectively. In particular, when cRGDyK-SS-CPLHSPT was treated at a concentration of 75 μM or more, the survival rate of tumor cells was less than 50%. On the other hand, in the case of cRGDyK-Asp-CPLHSpT peptide in which PLHSpT and RGD were not linked to Asp, the survival rate of HeLa tumor cells was similar to that of the comparative group (PLHSpT). Therefore, it can be confirmed that the fusion peptide according to the present invention has excellent intracellular migration characteristics and cancer cell growth inhibition properties.

[Experimental Example 2] Morphological assay of cRGDyK-SS-CPLHSPT

Human glioma cells (U87MG cells) and human cervical cancer cells (HeLa cells) were cultured in a 96-well plate for 24 hours at a cell number of 1 x 10 ³ per well. As the culture medium, DMEM medium, 10% FBS and 1X penicillin / streptomycin solution were used. The cultured cells were washed with a culture solution containing no FBS and cRGDyK, PLHSpT, and cRGDyK-SS-CPLHSPT were added to each well at a concentration of 50 μM, respectively, and reacted for 24 hours. After the reaction, the cells were replaced with fresh culture medium, and each cell was observed with an optical microscope (IX81, Olympus Inc.). The results are shown in FIG. 3B. As shown in FIG. 3B, when treated with cRGDyK-S-S-CPLHSPT ( 1 in FIG. 3B), the U87MG and HeLa tumor cells were rapidly damaged and the morphology of the cells It shows the stage of death. On the other hand, when treated with cRGDyK and PLHSpT, no morphology of cell death was observed.

[Experimental Example 3] Evaluation of intracellular delivery efficiency of fluorescent substance-bound cRGDyK-SS-CPLHSpT (fluorescence microscope)

Human cervical cancer cells (HeLa cells) were cultured in 24 wells at a cell number of 5 x 10 ⁴ for 24 hours. As the culture medium, DMEM or RPMI1640 medium, 10% FBS and 1X penicillin / streptomycin solution were used. On the other hand, FITC -PLHSpT , an optical probe (green), was obtained by treating PLHSpT and cRGDyK -S-S-CPLHSpT with a fluorescent substance (FITC, 6- [fluorescein- FITC-cRGDyK-SS-CPLHSpT. The cultured cells were treated with 50 [mu] M of FITC-PLHSpT and FITC-cRGDyK-SS-CPLHSpT, respectively, and reacted for 12 hours. After the reaction, the cells were replaced with fresh culture medium, and each cell was observed with a fluorescence microscope (IX81, Olympus Inc.). The results are shown in FIG. 3c. The cell nuclei of the cultured cells were stained with DAPI (4 ', 6-diamidino-2-phenylindole, blue) and observed with a fluorescence microscope (IX81, Olympus Inc.) Respectively. As shown in FIG. 3C, PLHSpT was not uptake into HeLa tumor cells, whereas fusion cRGDyK-SS-CPLHSpT (1 in Figure 3c) significantly increased uptake into HeLa tumor cells. From the above results, it can be confirmed that the intracellular uptake capacity of PLHSpT is increased due to the binding of cRGDyK.

[Experimental Example 4] Inhibitory effect of cRGDyK-SS-CPLHSpT on Plk-1 kinase

Human glial cells (U87MG cells) and human cervical cancer cells (HeLa cells) were cultured in 6 wells for 24 hours at a cell number of 5 x 10 ⁴ per well. As the culture medium, DMEM or RPMI1640 medium, 10% FBS and 1X penicillin / streptomycin solution were used. After the incubation, PLHSpT and cRGDyK-SS-CPLHSpT were added to each well at a concentration of 25 μM (U87MG) and 50 μM (HeLa cell), respectively, and reacted for 24 hours. The reaction solution was rinsed three times with PBS (phosphate buffer saline) buffer, and the effect of inhibiting the kinase (A450: 1: 100) was measured using a Plk-1 kinase 1 assay / inhibitor screening kit (MBL Inc., Japan) Absorbance at 450 nm) was measured, and the results are shown in Figs. 4A and 4B. As shown in Fig. 4A, in the human U87MG and HeLa cancer cells, Plk-1 kinase activity was significantly inhibited when treated with cRGDyK-SS-CPLHSpT, as compared with the case where PLHSpT was treated. In addition, as shown in FIG. 4B, when human HeLa and U87MG cancer cells were treated with cRGDyK-SS-CPLHSpT, Plk-1 kinase activity decreased with time, and remained constant after 24 hours.

[Experimental Example 5] Effect of cRGDyK-SS-CPLHSpT on cell division

Human glioblastoma cells (U87MG cells) and human cervical cancer cell lines (HeLa cells) were cultured in 6 wells for 24 hours at a cell number of 5 x 10 ⁴ per well. As the culture medium, DMEM or RPMI1640 medium, 10% FBS and 1X penicillin / streptomycin solution were used. The cultured cells were treated with 50 [mu] M of FITC-PLHSpT and FITC-cRGDyK-SS-CPLHSpT, respectively, and reacted for 12 hours. The reaction solution was washed with PBS (phosphate buffer saline) three times, fixed with 4% PFA (paraformaldehyde) for 30 minutes, and then washed three times with PBS buffer. Next, the reaction solution was stained with a Plk-1 antibody, a DAPI solution and a propidium iodide (PI) solution, washed with water and then subjected to fluorescence microscopy (IX81, Olympus Inc.) The results are shown in FIG. 4C. As shown in Fig. 4C, cells treated with cRGDyK-SS-CPLHSPT have multipolar spindles and misaligned chromosomes, and by continuing the division of the gene during the cell division, . Therefore, cRGDyK-SS-CPLHSPT inhibits Plk-1 activity, thereby blocking signal transduction involved in cell proliferation and inhibiting proliferation of cancer cells.

[Experimental Example 6] Effect of cRGDyK-SS-CPLHSpT on apoptosis

(1) Western blot assay

Human platelets (U87MG cells) and human cervical cancer cells (HeLa cells) were cultured in 6 wells for 24 hours at a cell number of 2.5 x 10 ⁴ per well. As the culture medium, DMEM or RPMI1640 medium, 10% FBS and 1X penicillin / streptomycin solution were used. Cultured cells were treated with cRGDyK, PLHSpT and cRGDyK-SS-CPLHSpT, respectively, at a concentration of 10 [mu] M, and reacted for 24 hours and 48 hours. (ADP-ribose) polymerase (PARP), which is a cytotoxic factor, in each tumor cell to confirm the apoptosis of the tumor cells treated with each of the peptides. The cleavage pattern was confirmed. Specifically, proteins were extracted from cultured tumor cells using PRO-PREP Protein Extraction Solution (iNtRON Biotech., Inc., Korea), and then the extracted proteins were subjected to gel electrophoresis, and polyvinylidene fluoride (ADP-ribose) polymerase (PARP) was detected using a polyvinylidene fluoride (PVDF) membrane and anti-PARP (anti-poly (ADP-ribose) The results are shown in FIG. 5A. Since the cell death factor PARP becomes cleaved PARP in the cell death process, the degree of apoptosis can be confirmed by quantifying the isolated PARP (PARP phosphorylation rate). As shown in Fig. 5A, the cleavability of PARP was significantly increased in tumor cells (U87MG and HeLa) treated with cRGDyK-SS-CPLHSpT ( 1 in Fig. 5A) as compared to tumor cells treated with PLHSpT . Especially, in the case of HeLa cells, when the reaction was carried out for 48 hours, the degree of separation of PARP was increased as compared with the case of reacting for 24 hours. Thus, apoptosis of U87MG and HeLa cells treated with cRGDyK-SS-CPLHSpT was confirmed by cleavage of PARP proportional to time course.

(2) FACS (fluorescence-activated cell sorting) analysis

Human cervical cancer cells (HeLa cells) were cultured in 6 wells at a cell number of 5 x 10 ⁴ per well for 24 hours. As the culture medium, DMEM or RPMI1640 medium, 10% FBS and 1X penicillin / streptomycin solution were used. The cultured cells were treated with FITC-cRGDyK, FITC-PLHSpT and FITC-cRGDyK-SS-CPLHSpT at a concentration of 50 μM, respectively, and reacted for 24 hours and 48 hours. After the reaction, cells were detached, washed three times with PBS, and fixed with 4% PFA (paraformaldehyde) for 30 minutes. Next, FACS analysis (FACS analysis) was performed using FACS (FACSCalibur, Becton, Dickinson and Company, USA), and the result is shown in FIG. 5B. In FIG. 5B, apoptosis was measured by annexin V and PI staining, and PBS was used as a control. As shown in FIG. 5B, the degree of apoptosis of tumor cells was quantified by FACS analysis. As a result, the degree of apoptosis was remarkably increased after 24 hours of reaction.

[Experimental Example 7] Anticancer effect of cRGDyK-SS-CPLHSpT in the body

(1) 2 x 10 ⁷ human cervical cancer cells (HeLa cells) were injected into the epidermis of Balb / c nude mice to induce cancer. PLHSpT and cRGDyK-SS-CPLHSpT were injected into the mouse tail vein for 15 days at 3-day intervals at a concentration of 4 mg / kg when the width and height of the induced cancer were 7 mm respectively, The results are shown in FIG. 6A, and a photograph of the tumor 19 days after the start of observation is shown in FIG. 6B. As shown in Fig. 6A, the growth of tumor tissues was significantly inhibited by the injection of cRGDyK-SS-CPLHSpT ( 1 in Fig. 6A), whereas in the control group in which nothing was treated and in the comparative group in which PLHSpT was treated, Have grown steadily. In addition, as shown in Fig. 6B, the tumor size of cRGDyK-SS-CPLHSpT ( 1 in Fig. 6A) -treated mice was inhibited by 5 times as much as the tumor size of mice treated with PLHSpT peptide. After 19 days, tumor tissues were cut to a thickness of 5 탆 and tumor tissues were stained according to the hematoxylin and Eosin (H & E) staining protocol. The results are shown in Fig. 6C. As shown in FIG. 6C, cRGDyK-SS-CPLHSpT inhibited tumor cell proliferation and promoted apoptosis, while tumor cells treated with PLHSpT continued to grow as with controls.

(2) In order to confirm the apoptosis of the mouse tumor cells, the tumor tissues were green (detected by apoptosis) with FITC-Annexin V Apoptosis Detection Kit (Sigma-Aldrich, USA) In order to identify the DNA, the tumor tissue was red colored with propidium iodide (PI, detection of DNA of the nucleus), and the result is shown in FIG. In FIG. 7, non-apoptotic cells were shown to be weak or absent in green and red fluorescence, and early apoptotic cells displayed weak orange or red color (weak or no green fluorescence) Late apoptotic cells and necrotic cells are represented by red fluorescence only. As shown in FIG. 7, in the control and PLHSpT treated tissues, the Annexin V positive portion was very small whereas in the tissue treated with the fusion peptide of the present invention, the Annexin V positive portion was very large (red or orange ), It can be seen that the fusion peptide of the present invention plays an important role in cell death.

[Experimental Example 8] In vitro and in vivo tests of radioactive tracer

(1) In vitro test

Example 2 ⁶⁸ Ga- labeled radiotracer prepared in (a peptide labeled with a ^{radiation, 68 Ga-NOTA-cRGDyK-} SS-CPLHSpT) using for diagnosing tumors, α _v β ₃ integrin-positive (integrin- positive human hybridoma (U87MG) cells and human cervical cancer (HeLa) cells were used. HeLa cells were cultured in DMEM medium containing 10% FBS, 50 mg / mL penicillin / streptomycin and glutamine, and U87MG cells were cultured in RPMI 1640 medium containing 10% FBS and 50 mg / mL penicillin / streptomycin Lt; / RTI > The cells were added to a 24-well plate at a concentration of 1 x 10 ⁵ cells / well and allowed to stand for 30, 60 and 120 minutes with the radioactive tracer, and then the in vitro cells of the radioactive tracer The cell binding affinity was evaluated, and the results are shown in FIG. 8A. The radioactivity of the cells was measured with a NaI (Tl) gamma counter.

As shown in Fig. 8A, in HeLa cancer cells, the tumor uptake rates at 30, 60, and 120 minutes were 1.51 + 0.03, 2.10 + 0.13, and 3.10 + 0.20%, respectively, of the total absorbance. For U87MG cancer cells, the tumor uptake rates at 30, 60, and 120 min were 1.49 0.10, 1.35 0.22, and 2.82 0.15%, respectively, of the total absorbance. As shown in Fig. 8A, the uptake rate of the radioactive tracer in cancer cells increases with time, which means that the radioactive tracer specifically binds to the [alpha] _{v [} beta] ₃ integrin receptor.

(2) In vivo test

In tumor-bearing mice, 1 x 10 ⁶ human brain glioma cancer cells (U87MG cells) were subcutaneously injected into Balb / c nude mice to obtain PET images of the radioactive tracker. After 3 weeks, 7.18 MBq of radioactive tracer (68Ga-NOTA-cRGDyK-SS-CPLHSpT) was injected into the tail vein of the mice and PET scanning was performed for 30 minutes, 60 minutes and 120 minutes after the injection of the radioactive tracer, The in vivo 68Ga-NOTA-cRGDyK-SS-CPLHSpT radioactive tracer profile (PET image) was obtained and the results are shown in Figure 8b (arrow indicates U87MG tumor). As shown in Figure 8b, the uptake rate of the radioactive tracer was maximal in the liver and kidney after 1 hour post-irradiation tracer injection.

To confirm the biodistribution of the radioactive tracer in tumor mice, 1 x 10 ⁶ U87MG cells were subcutaneously injected into nude mice and 20 days after injection of U87MG cells, 3.7 MBq Were injected. Samples of organs (blood, bone, heart, lung, liver, kidney, spleen, tumor and brain) were obtained by sacrificing the mice at 30, 60 and 120 minutes after the injection of the radioactive tracker, Activity was measured, and the results are shown in FIG. 8C. The radioactivity was expressed as a percentage of the injected dose (ID) per gram of tissue. As shown in Figure 8c, in biodistribution studies, maximal absorption of radiolabeled proteins was observed in the liver and kidney.

The uptake rate of the radioactive tracer was slightly higher in the tumor tissues than in the other tissues, and was maximal one hour after the injection. Tumor radiotracer uptake was 1.02 ± 0.25, 3.40 ± 1.10, and 0.62 ± 0.05% ID / g at 30, 60, and 120 min after injection, and the tumor-to-tissue ratio of tumor to tumor ratio were 2.55, 3.03, and 1.81, respectively, at 30, 60 and 120 minutes after the irradiation of the tracer. From the above results, it can be confirmed that the in vivo distribution of the radiation tracer correlates with the PET analysis result.

The present invention relates to a polynucleotide encoding a polo-box domain of polo-like kinase 1 targeted peptide in In vivo tumor imaging in Biomaterials (Volume 33, Issue 29, Pages 6915-6925, journal homepage: www.elsevier.com/locate/biomaterials) , Filed on June 06, 2012, and published online, and the entire contents of which are incorporated herein by reference.

<110> Korea Basic Science Institute <120> Fused peptide type tumor inhibitor, tumor diagnosing agent and          method for preparing the same <130> PD10366 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> inhibitor for polo box domain of Polo-like kinase <220> <221> MOD_RES <222> (5) <223> PHOSPHORYLATION, <400> 1 Pro Leu His Ser Thr   1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide for cell internalization <220> <221> CHAIN <222> (1) <223> cyclic form <220> <221> SITE <222> (4) <223> D-Tyr <400> 2 Arg Gly Asp Tyr Lys   1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> tumor inhibitor <220> <221> DISULFID <222> (5) (6) <220> <221> CHAIN <222> (1) <223> cyclic form <220> <221> SITE <222> (4) <223> D-Tyrosine <220> <221> MOD_RES <222> (11) <223> PHOSPHORYLATION, <400> 3 Arg Gly Asp Tyr Lys Cys Pro Leu His Ser Thr   1 5 10

Claims (7)

As a tumor suppressor having the amino acid sequence of SEQ ID NO: 3 below,
[SEQ ID NO: 3]
cRGDyK-SS-CPLHSpT
The tumor suppressor of SEQ ID NO: 3 comprises a PLHSpT peptide (Pro-Leu-His-Ser-p-Thr, SEQ ID NO: 1, wherein p- Thr represents Phosphorylated (PO ₃ H ₂ ^- ) Thr) and cRGDyK Wherein the peptide is a fusion peptide linked to a cysteine (C) -blocked disulfide bond by a peptide (cyclic Arg-Gly-Asp-D-Tyr-Lys, SEQ ID NO: 2). delete The tumor suppressor according to claim 1, wherein the tumor is selected from the group consisting of human glioma subtype and cervical cancer. A tumor diagnostic agent comprising a peptide having an amino acid sequence of SEQ ID NO: 3 and a detectable label (label). 5. The oncology diagnostic agent of claim 4, wherein the detectable label is selected from the group consisting of a radioactive isotope and a fluorescent luminescent material. 5. The oncology diagnostic agent according to claim 4, wherein the peptide having the amino acid sequence of SEQ ID NO: 3 and the detectable label are bound through an intermediate compound. attaching N-succinimidyl-3- (2-pyridyldithio) -propionate (SPDP) to the lysine side chain of cRGDyK peptide to produce SPDP-activated cRGDyK; And
Comprising the step of binding said SPDP-activated cRGDyK to an N-terminal cysteine of a CPLHSpT peptide.

Images