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WO2018199607A1 - Biomolecule imaging method using aptamer - Google Patents

Biomolecule imaging method using aptamer Download PDF

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Publication number
WO2018199607A1
WO2018199607A1 PCT/KR2018/004770 KR2018004770W WO2018199607A1 WO 2018199607 A1 WO2018199607 A1 WO 2018199607A1 KR 2018004770 W KR2018004770 W KR 2018004770W WO 2018199607 A1 WO2018199607 A1 WO 2018199607A1
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aptamer
odn
seq
composition
erbb2
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French (fr)
Korean (ko)
Inventor
이중환
김종인
임종훈
이종욱
김진우
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Interoligo Corp
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Interoligo Corp
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Priority claimed from KR1020180046550A external-priority patent/KR102062115B1/en
Application filed by Interoligo Corp filed Critical Interoligo Corp
Priority to EP18791814.9A priority Critical patent/EP3636287B1/en
Priority to US16/608,645 priority patent/US11499156B2/en
Priority to JP2019557747A priority patent/JP7016544B2/en
Priority to CN201880027916.2A priority patent/CN110621351B/en
Publication of WO2018199607A1 publication Critical patent/WO2018199607A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a biomolecular imaging method using aptamers, and more particularly, to a method of obtaining an image by binding to a growth factor receptor 2 (HER2) expressing cell line using an aptamer labeled with an isotope.
  • HER2 growth factor receptor 2
  • aptamer's etymology comes from the Latin "aptus” meaning “just fit” and the Greek word “meros” meaning “part”. These aptamers are single-stranded nucleic acids consisting of 20 to 90 bases in length.
  • SELEX vitro artificial evolution
  • an aptamer discovery technique selects aptamers that are highly specific and affinity for the target molecule.
  • aptamers are considered to be very good reagents for measuring or finding the expression level of specific molecules they target.
  • Aptamers have advantages over antibodies in several respects, including low cost of production, ease of synthesis, low toxicity, no immune response, and no production in animal systems such as antibodies. And this is a relatively new reagent in the diagnostic field.
  • Aptamers are being developed for a wide variety of targets, such as thrombin, nucleolin, PSMA, TNC, and proteins of viral origin.
  • targets such as thrombin, nucleolin, PSMA, TNC, and proteins of viral origin.
  • the first FDA-approved VEFG target aptamer was developed in 2004 for the treatment of elderly macular degeneration.
  • a large number of aptamers are being developed at preclinical and clinical stages, and more trials are underway in terms of diagnosis and treatment.
  • HER2 is a very well known cancer gene that is increased or overexpressed by approximately 15-30% of breast cancers. And in many cancers, high rates of relapse and prognosis are factors associated with very poor outcomes.
  • the two signaling systems activated by HER2 have a MAPK pathway that promotes cell proliferation and a PI3K-AKT pathway that promotes cancer cell survival. Therefore, it is considered as a very suitable target for application to cancer treatment.
  • transtuzumab and pertuzummab which target HER2 are well known therapeutic single antibodies that are currently commercially available.
  • DNA / RNA aptamers targeting HER2 have been unearthed through traditional SELEX and cell-based SELEX. And recently, an example of pharmacologically utilizing the cancer-inhibiting properties of such HER2 aptamers has been reported.
  • molecular imaging can visualize biochemical events in real time at the cellular and molecular level of living cells, tissues or intact objects in a non-invasive way.
  • Aptamers modified with magnetic nanomaterials or fluorescent materials can serve as good materials for targeted fluorescence imaging or magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • Several in vivo MRI studies have observed effective targeting of cancer in mice with cancer.
  • PET clearly has advantages in diagnostics over anatomical techniques such as computed tomography (CT) and MRI.
  • CT computed tomography
  • PET has widespread application in basic research and preclinical applications. For example, PET can be used to verify new radiotherapy assays, efficacy of new therapeutic agents, and upper body distribution of drugs.
  • PET is a representative molecular imaging device that can detect biochemical changes in the molecular target level of living organisms using radiopharmaceuticals and is very sensitive and is used in a wide range of fields including basic science and preclinical.
  • Targeting cancer with aptamers is a recent molecular imaging technique that has been used by Hicke and other researchers in molecular imaging. They covalently bound 99m TC to an aptamer called TTA1, which binds to the extracellular matrix protein tenascin-C, and imaged cancer using gamma-camera in vivo. Later, imaging with PET was implemented by another team.
  • Aptamers are a class of nucleic acids that have high specificity and affinity for target molecules.
  • An object of the present invention is to obtain a molecular image in vivo using an aptamer labeled with radioisotopes or fluorescent dyes.
  • Figure 1 shows a schematic of the mechanism of radioisotopes or fluorescently labeled ERBB2 aptamers.
  • HER2 aptamer labeled with radioisotopes or fluorescent dyes was used for in vivo imaging.
  • ERBB2 aptamers hardly bind to MDA-MB231 cell lines that do not express HER2, but have very high affinity for BT474, a HER2 expressing cell line.
  • the aptamers were observed to bind in HER2-expressing breast cancer cell lines and only minimal binding in HER2 unexpressed cells.
  • Positron tomography molecular imaging of mice implanted with BT474 cancer cell line in vivo significantly increased the uptake of HER2-specific ERBB2 aptamers labeled 18 F into cancer cells.
  • ERBB2 aptamers can bind preferentially to HER2 expressed breast cancer cell lines both in vitro and in vivo because it is possible to recognize HER2 structures on the cell surface.
  • ERBB2 aptamers labeled with radioactive isotopes or fluorescent dyes, such as 18 F, can recognize HER2 expression in human breast cancer cells and enable appropriate visualization. These results suggest potential applications for or treatment of isotopes or fluorescent dye-labeled ERBB2 aptamers for HER2-positive breast cancer cells.
  • FIG. 1 is a schematic diagram of the mechanism of the radiation or fluorescent label ERBB2 aptamer.
  • Figure 3 shows cholesteryl- [AP001-24] -ODN-idT, cholesteryl- [AP001-24] -ODN aptamer and fluorescent label cODN (cODN-Cy5) at 50, 55 and 60 degrees temperatures.
  • cODN-Cy5 fluorescent label
  • Figure 4 shows cholesteryl- [AP001-24] -ODN-idT, cholesteryl- [AP001-24] -ODN, PEGylated- [AP001-25] -ODN-idT, PEGylated- [AP001-
  • the complementary base pairing between 25] -ODN aptamer and fluorescent label cODN (cODN-Cy5) was confirmed by agarose gel.
  • FIG. 5 shows confocal image results of R- [ERBB2 aptamer] -X- hy (bp) -Cy5 for KPL4, N87, and SK-BR cell lines. Confocal microscopy images of [AP001-24] -hy (bp) -Cy5 and [AP001-25] -hy (bp) -Cy5 aptamers in HER2-positive cell lines.
  • FIG. 6 shows the results of FACS analysis of R- [ERBB2 aptamer] -X- hy (bp) -Cy5 for KPL4, N87, and SK-BR cell lines.
  • FIG. 10 shows microPET images of cholesteryl- [AP001-24] -i dT- hy (bp) -LF 18 .
  • Figure 11 is PEGylated- [AP001-24] - hy (bp ) is a result of the microPET image -LF 18.
  • FIG. 13 shows [AP001-24] -hy (bp) -LF 18 , [AP001-24] -idT-hy (bp) -LF 18 , cholesteryl- [AP001-24] -hy in mice bearing KPL4 cancer. Comparative image of (bp) -LF 18 , cholesteryl- [AP001-24] -idT-hy (bp) -LF 18 .
  • FIG. 15 shows flow cytometry analysis of breast cancer cell lines using HER2 antibody and ERBB2 aptamer (AP001-25).
  • A Viscosity table shows BT474 from antibody and (HER2 positive cell line), MDA-MB231 (HER2 negative cell line). Indicates a fluorescence signal.
  • ERBB2 aptamer (AP001-25) red
  • control sequence blue
  • b Flow cytometry graphs in two cell lines using antibodies, ERBB2 aptamer (AP001-25) and negative controls.
  • 16 is a confocal microscopy image of selected ERBB2 aptamers (AP001-25) in HER2-positive cell lines.
  • Figure 17 in a mouse cancer embraces a BT474 18 F labeled ERBB2 aptamer ⁇ [AP001-25] - hy (bp ) -LF 18 ⁇ in a PET image representing the biometric.
  • Hy (bp) represents ODN / cODN hybridization by hybridization (base pairing).
  • the PET image in the living body representing - ⁇ hy (bp) 18 -LF [AP001-25] ⁇ Fig. 19 is in mice with normal HER2 positive and negative arm 18 F labeled ERBB2 aptamer.
  • HER2 overexpression BT474 cancer shows increased uptake when compared to
  • HER2 negative MDA-MB231 cancer shows increased uptake when compared to
  • HER2 negative MDA-MB231 cancer right armpit.
  • CCM Numbers per minute
  • amount injected per gram of cancer tissue % ID / g were calculated with an 18 F labeled ERBB2 aptamer.
  • the ERBB2 aptamer that specifically binds to the breast cancer related Her2 receptor used in the present invention has a base sequence of 5'-TCAGCCGCCAGCCAGTTC- [Core sequence] -GACCAGAGCACCACAGAG-3 'and the number 6 in the Core Sequence or n in the attached base sequence list. Represents NaptyldU.
  • radioisotopes such as 18 F, 32 P, 123 I, 89 Zr, 67 Ga, 201 Tl, 111 In- HER2 aptamer labeled with 111 or a fluorescent dye, for example Cy3, Cy5, Cy7, etc., was used for in vivo imaging.
  • an ERBB2 aptamer labeled with an isotope or a fluorescent dye was used to evaluate target specificity and clinical applicability for molecular imaging in vivo.
  • ERBB2 aptamers for human epidermal growth factor receptor 2 were labeled with 18 F-fluoride isotopes.
  • flow cytometry and confocal microscopy were compared with control aptamer.
  • Positron tomography of HER2-specific ERBB2 aptamers labeled with 18 F was used to obtain biomolecular images of mice transplanted with BT474 or KPL4 cells over time, respectively.
  • HER2 expressed human breast cancer cell lines BT474, KPL4, N87, and SK-BR-3 were used for in vitro and in vivo testing. And the experiment was conducted with another human breast cancer cell line MDA-MB231 as a control. All cell lines were purchased from ATCC and maintained in culture in MEM medium containing 10% FBS.
  • Cell lysates containing protease inhibitors were incubated for 30 minutes on ice to extract intracellular proteins.
  • the cell lysate thus obtained was purified by centrifugation at 4 ° C. for 20 minutes. Protein quantification was performed using the Bradford method and 30 ⁇ g of protein extract from each sample was isolated by electrophoresis with 10% SDS-PAGE. Then, it was transferred to the nitrocellulose membrane and sensitized to the x-ray film by ECL using a HER2 antibody and a control beta-action antibody as a probe.
  • AP001-24 the ERBB2 aptamer, has a binding force (Kd) of 3.1 nM to the target and AP001-25 has 0.9 nM.
  • ODN 5'-CAGCCACACCACCAG-3 '
  • AP001-24 a fully matching sequence at 3' of ERBB2 aptamers ⁇ [AP001-24] and [AP001-25] ⁇
  • the aptamer synthesis process was synthesized using the solid phase synthesis method using phosphoramidite coupling reaction, and the reaction was carried out for 5 hours at 70 ° C. in t -butylamine: methanol: water (1: 1: 2 v / v / v) Through cleavage and deprotection process, the whole aptamer was obtained and dried. Synthetic aptamer was separated by HPLC [C18 column (Waters, Xbridge OST C18 10x50mm, 260nm)] and then molecular weight was measured using an ESI MS mass spectrometer (Qtrap2000, ABI).
  • the 11th aptamer (SEQ ID NO: 11) of Table 1 corresponds to AP001-24.
  • the 12th aptamer (SEQ ID NO: 7) in Table 1 corresponds to AP001-25.
  • CAG-3 ' ⁇ each aptamer-ODN ⁇ of (SEQ ID NO: 1 to 35) in Table 1 was synthesized by the ⁇ [AP001-24] -ODN ⁇ synthesis method described above.
  • Cy5-labeled ERBB2 aptamer ⁇ R- [ERBB2 aptamer] -X-hy (bp) -Cy5 ⁇ , was made in the following manner.
  • 18 F-labeled cODN synthesis centered on a previously reported procedure (Ref. 24). No-carrier-added 18F-fluoride ion was generated using a synthesizer (Tracerlab FXFN, GE Healthcare, Milwaukee, WI, USA), followed by reaction with mesylate (10 minutes at 100 ° C) and 18 F-fluoro-PEG -azide (18F-FPA) was purified using HPLC.
  • the synthesized 18 F-labeled cODN (cODN-LF 18 ) was purified by HPLC H (Xbridge OST C18 10 ⁇ 50mm, eluent acetonitrile / 0.1 M TEAA 5: 95-95: 5 over 20 minutes, flow rate: 5 mL / min, purification using UV (254 nm).
  • the F 18 radioisotope label ERBB2 aptamer ⁇ R- [ERBB2 aptamer] -X-hy (bp) -LF 18 ⁇ , was made in the following manner.
  • BT474, KPL4, N87, SK-BR-3, and MDA-MB231 cell lines were aliquoted onto the coverslip and incubated overnight. Giving carefully wash time approximately 80% grow up a fluorescence labeled ERBB2 aptamer ⁇ R- [ERBb2 aptamer] - hy (bp) -Cy5 ⁇ were incubated treated at a concentration of 250nM. After incubation, the cells were washed carefully and a medium containing DAPI was mounted on the slide. Fluorescence was observed with an LSM700 confocal microscope. The microscopic setting was an excitation with a 488nm laser for FITC observation, BP490-555 for emission, and a 6640nm laser for Texas red with LP640 filter.
  • ERBB2 overexpressing breast cancer cell lines KPL4, N87 and SK-BR-3 were dispensed onto the coverslip and incubated overnight.
  • the cells were washed carefully and incubated by processing a sample in which Cy5 fluorescence-labeled ODN was bound to ERBb2 aptamer using complementary base pairing. After incubation, the cells were washed carefully and a medium containing DAPI was mounted on the slide. Fluorescence was observed with an LSM700 confocal microscope.
  • ERBB2 aptamer The specificity of ERBB2 aptamer was verified by fluorescence activated cell separation using flow cytometry (BD Biosciences). BT474, KPL4, N87, SK-BR-3, or MDA-MB231 cancer cell lines were cultured to pass up to 80% by passage of appropriate numbers in Petri dishes. The cells were trypsinized and washed with PBS, and the fluorescently labeled ODN was bound to ERBB2 aptamer as a complementary base by temperature. The completed sample was treated with the cells. ERBB2 aptamer ⁇ R- [ERBb2 aptamer] -hy (bp) -Cy5 ⁇ and an antibody containing 1% FBS as a control were each treated at 4 ° C. for 30 minutes. The treated samples were washed and then bound ERBB2 aptamer was measured and analyzed by fluorescence-activated cell separation.
  • 17 ⁇ -estradiol pellets were implanted subcutaneously in 4 week old Balb / c nude mice to release estrogens in cancerous areas. After a few days 7X10 6 per mouse BT474 or KPL4 human breast cancer cell lines were implanted subcutaneously in large numbers. After three weeks of cancer, the growth of the cancer was measured by a caliper.
  • mice Balb / C nude mice were implanted subcutaneously with 1 ⁇ 10 5 human breast cancer cell line KPL4 cells in the right shoulder. It was then induced to develop cancer.
  • mice were acquired using Inveon microPET (Siemens, Knoxville, TN, USA) scanner for 60 minutes from 60 minutes after F 18 radioisotope labeled ERBB2 aptamer injection. Respiratory anesthesia with 2% Isoflurane upon F 18 radioisotope labeled ERBB2 aptamer injection followed by injection of 7.4 MBq of F 18 radioisotope labeled ERBB2 aptamer into the tail vein of mice. The obtained listmode data was transformed into sinograms and reconstructed with a 3D Ordered Subset Expectation Maximization (OSEM) algorithm and analyzed using ASIpro (Concorde Microsystems Inc, Knoxville, TN).
  • OEM Ordered Subset Expectation Maximization
  • F 18 radioactive isotopic label ERBB2 The aptamer was injected intravenously into tumor-grown mice by human breast cancer cell injection, and then PET was performed using Siemens inveopn PET. The amount injected was 13.7 ⁇ 1.1 MBq (370 ⁇ 30 uCi) and the dynamic PET study was performed for 30 minutes according to 10 1 minute images and 4 5 minute imaging protocols. Two stationary studies were performed for 10, 60, 90 and 120 minutes after infusion. Partial quantification of PET signals was performed using AMIDE software. Images were implemented using a false-color scale proportional to the tissue concentration (% ID / g) of the positron labeled probe. Red indicates the highest concentration, and yellow, green, and blue gradually match the lower concentrations.
  • PET images are shown in FIGS. 7-13.
  • the HER2 antibody was shown to bind very specifically to the HER2-positive BT474 cell line using flow cytometry.
  • ERBB2 aptamers are very weak in the MDA-MB231 cell line but strongly bind in the BT474 cell line.
  • no binding to any cell line was seen in random nucleic acid oligos.
  • ERBB2 aptamer Cell binding of ERBB2 aptamer was further evaluated by confocal microscopy (FIG. 16).
  • BT474 HER2-positive breast cancer cell lines were treated with aptamers.
  • the ERbB2 aptamer was labeled with fluorescence and fluorescence was observed on the cell surface, and it was confirmed that the HER2 structure was present on the surface of these cells.
  • the fluorescence represented by the aptamer was observed along the cell membrane, and no fluorescence signal was observed in the negative control MDA-MB231 cell line. Thus, it was observed that this ERBB2 aptamer can bind to HER2-positive breast cancer cell lines and minimal binding to HER2 negative cells.
  • ERBB2 aptamers ⁇ [AP001-24] and [AP001-25] ⁇ Treatment of ERBB2 aptamers ⁇ [AP001-24] and [AP001-25] ⁇ with complementary base pairs with fluorescently labeled ODN was performed on KPL4, N87 and SK-BR-3 breast cancer cell lines in the same manner as the experiments conducted above. After fluorescence was observed under a confocal microscope. Both ERBB2 aptamers were found to bind well to breast cancer cell lines, [AP001-24] was observed at the cell surface along the cell membrane, and [AP001-25] was also observed inside the cell.
  • Biodistribution was validated in mice bearing cancer one hour after injection of ERBB2 aptamer labeled with 18 F. After sacrificing the animals, radioactivity levels in each tissue, including cancer, were measured using a gamma meter to express% ID / g (FIG. 18). It is also shown in Table 5 below.
  • the intake of 18 F labeled ERBB2 aptamers was 0.62 ⁇ 0.04 per hour.
  • Studies on biodistribution show that the two major release pathways of the 18 F-labeled ERBB2 aptamer are the kidneys and intestines.
  • FIG. 19 shows images showing 18 F labeled ERBb2 aptamers in mice bearing HER2 positive and negative cancers.
  • HER2 overexpression BT474 cancer shows high isotope uptake compared to HER2 negative MSA-MB231 cancer.
  • the total activity (nCi) in VOI voxel- or volume-of-interest
  • nCi total activity in VOI
  • BT474 cancer cells (upper row) are observed to stain the cell membrane more strongly against HER2 compared to the MDA-MB231 cell line (lower row).
  • HER2 target ERBB2 aptamer was successfully PET imaged in vivo.
  • the present invention is the first case of HER2 target PET imaging using specific ERBB2 aptamer. PET images from mice with BT474 cancer were confirmed by the ERBB2 aptamer to recognize HER2 in vivo and show the cancer relatively clearly. These results could potentially be applied to the radiotherapy-labeled ERBB2 aptamer or to determine the treatment method for targeted treatment of HER2-positive breast cancer cell lines.
  • Doggrell SA Pegaptanib: the first antiangiogenic agent approved for neovascular macular degeneration. Expert opinion on pharmacotherapy. 2005; 6: 1421-3.
  • Tan M Yu D. Molecular mechanisms of erb B2-mediated breast cancer chemoresistance. Advances in experimental medicine and biology. 2007; 608: 119-29.

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Abstract

The present invention relates to a composition for imaging a tumorous disease region, comprising a fluorescence- or radioactive isotope-labeled ERBB2 aptamer, wherein an ERBB2 aptamer labeled with a radioactive isotope or a phosphorescent dye is used to image a tumorous disease region in vivo.

Description

압타머를 이용한 생체 분자 영상화 방법Biomolecular Imaging Using Aptamer

본 발명은 압타머를 이용한 생체 분자 영상화 방법에 관한 것으로, 상세하게는 동위원소로 표지된 압타머를 이용하여 성장인자 수용체2(HER2) 발현 세포주에 결합시켜 영상을 얻는 방법에 관한 것이다. The present invention relates to a biomolecular imaging method using aptamers, and more particularly, to a method of obtaining an image by binding to a growth factor receptor 2 (HER2) expressing cell line using an aptamer labeled with an isotope.

압타머의 어원은 "꼭 들어맞다"라는 의미의 라틴어인 "aptus", "부분"이라는 의미의 그리스어인 "meros"에서 유래가 되었다. 이러한 압타머는 단일가닥 핵산으로 길이가 20에서 90개 정도의 염기 서열로 이루어져 있다. 보통 압타머 발굴 기술인 시험관 내 인공진화법(SELEX)을 통하여 표적분자에 매우 특이적이고 친화도가 높은 압타머를 선별한다. 따라서 압타머는 그들이 표적으로 하는 특정 분자의 발현도를 측정하거나 찾아내는데 아주 좋은 시약으로 간주되고 있다. 압타머는 생산비용에서의 저렴함, 합성이 용이한점, 낮은 독성, 면역반응을 불러 일으키지 않는 점 그리고 항체와 같이 동물 시스템에서 만들지 않는 등의 몇 가지 측면에서 항체와 비교하여 장점을 가지고 있다. 그리고 이것은 진단분야에서 비교적 새롭게 개발된 시약이다. 여러 가지 표적들, thrombin, nucleolin, PSMA, TNC 그리고 바이러스에 기원된 단백질과 같이 매우 다양한 표적에 대한 압타머들이 개발이 되고 있다. 치료분야에서는 2004년 최초로 노인 황반변성 치료제로 FDA에 승인된 VEFG 표적 압타머가 개발이 되었다. 현재에서는 매우 많은 종류의 압타머들이 전임상과 임상단계에서 개발중에 있고 더 많은 시험들이 진단 및 치료에 관련해서 진행중에 있다. The aptamer's etymology comes from the Latin "aptus" meaning "just fit" and the Greek word "meros" meaning "part". These aptamers are single-stranded nucleic acids consisting of 20 to 90 bases in length. In vitro artificial evolution (SELEX), an aptamer discovery technique, selects aptamers that are highly specific and affinity for the target molecule. Thus, aptamers are considered to be very good reagents for measuring or finding the expression level of specific molecules they target. Aptamers have advantages over antibodies in several respects, including low cost of production, ease of synthesis, low toxicity, no immune response, and no production in animal systems such as antibodies. And this is a relatively new reagent in the diagnostic field. Aptamers are being developed for a wide variety of targets, such as thrombin, nucleolin, PSMA, TNC, and proteins of viral origin. In the field of therapy, the first FDA-approved VEFG target aptamer was developed in 2004 for the treatment of elderly macular degeneration. At present, a large number of aptamers are being developed at preclinical and clinical stages, and more trials are underway in terms of diagnosis and treatment.

HER2는 매우 잘 알려진 암유전자로 대략 유방암의 15~30% 정도로 증가되거나 과발현되어 있다. 그리고 여러 암에서 높은 비율의 재발률과 예후가 매우 안 좋은 것과 관련된 인자이다. HER2에 의해 활성화되는 두 가지의 신호전달 체계는 세포증식을 촉진시키는 MAPK 경로와 암세포의 생존을 증진시키는 PI3K-AKT 경로가 존재한다. 따라서 암치료에 적용하는데 매우 적합한 표적으로 간주된다. 여기에 현재 상용된 잘 알려진 치료 단일 항체로 HER2를 표적화하는 트랜스투주맙(transtuzumab)과 퍼투주맙(pertuzummab)이 존재하는데 임상에서 효과적이라고 판명되었다. 이전에 HER2를 표적으로 하는 몇 가지의 DNA/RNA 압타머가 전통적인 방법의 SELEX와 세포를 기반으로 하는 SELEX를 통하여 발굴이 되었다. 그리고 그러한 HER2 압타머의 암을 저해하는 특성을 약학적으로 활용하는 예가 최근에 보고되었다. HER2 is a very well known cancer gene that is increased or overexpressed by approximately 15-30% of breast cancers. And in many cancers, high rates of relapse and prognosis are factors associated with very poor outcomes. The two signaling systems activated by HER2 have a MAPK pathway that promotes cell proliferation and a PI3K-AKT pathway that promotes cancer cell survival. Therefore, it is considered as a very suitable target for application to cancer treatment. Herein, transtuzumab and pertuzummab, which target HER2, are well known therapeutic single antibodies that are currently commercially available. Previously, several DNA / RNA aptamers targeting HER2 have been unearthed through traditional SELEX and cell-based SELEX. And recently, an example of pharmacologically utilizing the cancer-inhibiting properties of such HER2 aptamers has been reported.

한편, 분자 영상은 비침습적 방법으로 살아있는 세포나 조직 또는 손상을 가하지 않는 물체를 세포적 분자적 수준에서 생화학적 사건을 실시간으로 시각화할 수 있다. 자성 나노물질이나 형광소재로 변형된 압타머는 표적화 형광 영상화나 magnetic resonance imaging (MRI)에서 좋은 소재로 제공될 수 있다. 몇 생체 내 MRI 연구들에서 암이 있는 마우스에서 암을 효과적으로 표적화하는 것이 관찰되었다. 그러나 대사변화가 보통 해부학적 변화 전에 발생하기 때문에 PET은 분명하게 computed tomography (CT) 와 MRI 같은 해부학적 기술보다 진단에서 유리한 점이 있다. 임상활용에서 PET는 기초 연구와 전임상 분야에서 광범위한 응용이 되고 있다. 예를 들면 PET는 새로운 방사선치료제 분석, 새로운 치료제의 효능 그리고 약제의 상체분포를 검증하는데 사용될 수 있다. PET이 가지고 있는 강점으로 탐침 깊이, 탁월한 민감도, 정량적 자료 그리고 전임상에서 임상까지의 전환성에 있다. 즉, PET은 대표적인 분자영상기기로 방사선 의약품을 이용하여 살아있는 생체의 분자표적 수준에서의 생화학적 변화를 감지할 수 있고 굉장히 민감도가 높아 기초과학이나 전임상 영역을 비롯한 광범위한 분야에서 이용되고 있다. 압타머를 이용한 암 표적화는 최근에 생겨난 분자 영상기술로 Hicke을 비롯한 여러 연구자들에 의해 분자 영상에서 압타머가 사용된 사례가 있다. 그들은 세포외질 단백질인 tenascin-C에 결합하는 TTA1이라는 압타머에 99mTC를 공유결합하여 생체 내에서 gamma-camera를 이용하여 암을 영상화하였다. 차후에 PET을 이용한 영상화도 다른 팀에 의해 구현되었다. On the other hand, molecular imaging can visualize biochemical events in real time at the cellular and molecular level of living cells, tissues or intact objects in a non-invasive way. Aptamers modified with magnetic nanomaterials or fluorescent materials can serve as good materials for targeted fluorescence imaging or magnetic resonance imaging (MRI). Several in vivo MRI studies have observed effective targeting of cancer in mice with cancer. However, since metabolic changes usually occur before anatomical changes, PET clearly has advantages in diagnostics over anatomical techniques such as computed tomography (CT) and MRI. In clinical applications, PET has widespread application in basic research and preclinical applications. For example, PET can be used to verify new radiotherapy assays, efficacy of new therapeutic agents, and upper body distribution of drugs. The strengths of PET include probe depth, excellent sensitivity, quantitative data and the transition from preclinical to clinical. In other words, PET is a representative molecular imaging device that can detect biochemical changes in the molecular target level of living organisms using radiopharmaceuticals and is very sensitive and is used in a wide range of fields including basic science and preclinical. Targeting cancer with aptamers is a recent molecular imaging technique that has been used by Hicke and other researchers in molecular imaging. They covalently bound 99m TC to an aptamer called TTA1, which binds to the extracellular matrix protein tenascin-C, and imaged cancer using gamma-camera in vivo. Later, imaging with PET was implemented by another team.

그러나 HER2 특이 ERBB2 압타머를 이용한 PET 영상화에 대한 구현은 아직까지 알려진 바가 없다. However, the implementation of PET imaging using HER2-specific ERBB2 aptamers is not known yet.

압타머는 핵산류의 일종으로 표적분자에 높은 특이성과 친화도를 갖는 물질이다. 본 발명은 방사성 동위원소나 형광염료로 표지된 압타머를 이용하여 생체내의 분자적 영상을 얻는 것을 목적으로 한다. Aptamers are a class of nucleic acids that have high specificity and affinity for target molecules. An object of the present invention is to obtain a molecular image in vivo using an aptamer labeled with radioisotopes or fluorescent dyes.

도 1에는 방사성 동위원소 또는 형광표지 ERBB2 압타머의 메카니즘 모식도를 나타내었다. Figure 1 shows a schematic of the mechanism of radioisotopes or fluorescently labeled ERBB2 aptamers.

본 발명에서는 방사성 동위원소나 형광염료로 표지된 HER2 압타머를 이용하여 생체 내 영상화를 위해 활용하였다. In the present invention, HER2 aptamer labeled with radioisotopes or fluorescent dyes was used for in vivo imaging.

유동세포계수법을 통한 분석에서 ERBB2 압타머는 HER2가 발현하지 않는 MDA-MB231세포주에는 거의 결합하지 않는 반면 HER2 발현 세포주인 BT474에는 매우 높은 친화력을 갖는 것으로 보여진다. 마찬가지로 공초점 현미경으로부터 얻어진 영상에서도 이 압타머는 HER2가 발현된 유방암 세포주에서 결합하는 것으로 관찰이 되고 HER2 미발현 세포에서는 최소한의 결합만 하는 것으로 확인되었다. 생체 내 BT474 암 세포주가 이식된 마우스에 대한 양전자 단층촬영 분자 영상에서 암세포로의 18F 이 표지된 HER2 특이 ERBB2 압타머의 섭취가 상당히 증가되었다. ERBB2 압타머는 시험관 및 생체 내 두 가지에서 모두 HER2가 발현된 유방암 세포주에 우선적으로 결합할 수 있고 이것은 세포 표면에 HER2 구조체를 인지함이 가능하기 때문이다. Flow cytometry analysis showed that ERBB2 aptamers hardly bind to MDA-MB231 cell lines that do not express HER2, but have very high affinity for BT474, a HER2 expressing cell line. Similarly, in the images obtained from confocal microscopy, the aptamers were observed to bind in HER2-expressing breast cancer cell lines and only minimal binding in HER2 unexpressed cells. Positron tomography molecular imaging of mice implanted with BT474 cancer cell line in vivo significantly increased the uptake of HER2-specific ERBB2 aptamers labeled 18 F into cancer cells. ERBB2 aptamers can bind preferentially to HER2 expressed breast cancer cell lines both in vitro and in vivo because it is possible to recognize HER2 structures on the cell surface.

18F 등 방사성 동위원소나 형광염료가 표지된 ERBB2 압타머는 인간 유방암 세포에서의 HER2 발현을 인지하여 적절한 시각화를 가능하게 할 수 있다. 이러한 결과들은 동위원소나 형광염료 표지 ERBB2 압타머가 HER2 양성 유방암 세포에 대한 표적 치료 적용이나 어떻게 치료할 것인가에 대한 잠재적 응용법을 제시한다. ERBB2 aptamers labeled with radioactive isotopes or fluorescent dyes, such as 18 F, can recognize HER2 expression in human breast cancer cells and enable appropriate visualization. These results suggest potential applications for or treatment of isotopes or fluorescent dye-labeled ERBB2 aptamers for HER2-positive breast cancer cells.

도 1은 방사선 또는 형광표지 ERBB2 압타머의 메카니즘 모식도.1 is a schematic diagram of the mechanism of the radiation or fluorescent label ERBB2 aptamer.

도 2는 R-[ERBB2 압타머]-ODN-X (R = H, 콜레스테롤, 또는 PEG, X = H, 또는 idT)와 cODN-Cy5의 혼성화(hybridization) 결과물인 R-[ERBB2 압타머]-X-hy(bp)-Cy5를 Typhoon FLA7000 3% 아가로오스 겔로 분석한 것이다.FIG. 2 shows R- [ERBB2 aptamer] -ODN-X (R = H, cholesterol, or PEG, X = H, or idT) as a result of hybridization of cODN-Cy5 with R- [ERBB2 aptamer]- X- hy (bp) -Cy5 was analyzed on Typhoon FLA7000 3% agarose gel.

도 3은 50도, 55도, 60도 온도에 따른 콜레스테릴-[AP001-24]-ODN-idT, 콜레스테릴-[AP001-24]-ODN 압타머와 형광 표지 cODN(cODN-Cy5) 사이의 상보적 염기 쌍합을 3% 아가로오스 겔로 확인한 결과 (검은색: 압타머, 붉은색: cODN-Cy5).Figure 3 shows cholesteryl- [AP001-24] -ODN-idT, cholesteryl- [AP001-24] -ODN aptamer and fluorescent label cODN (cODN-Cy5) at 50, 55 and 60 degrees temperatures. Complementary base pairs between were identified by 3% agarose gel (black: aptamer, red: cODN-Cy5).

도 4는 95도 가열한 콜레스테릴-[AP001-24]-ODN-idT, 콜레스테릴-[AP001-24]-ODN, PEGylated-[AP001-25]-ODN-idT, PEGylated-[AP001-25]-ODN 압타머와 형광 표지 cODN(cODN-Cy5) 사이의 상보적 염기 쌍합을 아가로오스 겔로 확인한 결과. Figure 4 shows cholesteryl- [AP001-24] -ODN-idT, cholesteryl- [AP001-24] -ODN, PEGylated- [AP001-25] -ODN-idT, PEGylated- [AP001- The complementary base pairing between 25] -ODN aptamer and fluorescent label cODN (cODN-Cy5) was confirmed by agarose gel.

도 5는 R-[ERBB2 압타머]-X-hy(bp)-Cy5를 KPL4, N87, 및 SK-BR 세포주에 대한 confocal image 결과이다. HER2 양성 세포주에서의 [AP001-24]-hy(bp)-Cy5와 [AP001-25]-hy(bp)-Cy5 압타머의 공초점 현미경 영상. (a) KPL4, HER2 양성 유방암 세포주에서 Cy5 표지 압타머로 변환, (b) N87 암세포주에서 같은 압타머의 처리 (c), SK-BR-3 암세포주에서 같은 압타머의 처리 (표지 DAPI:파란색, Cy5-압타머:붉은색).5 shows confocal image results of R- [ERBB2 aptamer] -X- hy (bp) -Cy5 for KPL4, N87, and SK-BR cell lines. Confocal microscopy images of [AP001-24] -hy (bp) -Cy5 and [AP001-25] -hy (bp) -Cy5 aptamers in HER2-positive cell lines. (a) conversion of KPL4, HER2-positive breast cancer cell line to Cy5 labeled aptamer, (b) treatment of the same aptamer in N87 cancer cell line, (c) treatment of the same aptamer in SK-BR-3 cancer cell line (labeled DAPI: blue , Cy5-aptamer: red).

도 6은 R-[ERBB2 압타머]-X-hy(bp)-Cy5를 KPL4, N87, 및 SK-BR 세포주에 대한 FACS analysis 결과이다.6 shows the results of FACS analysis of R- [ERBB2 aptamer] -X- hy (bp) -Cy5 for KPL4, N87, and SK-BR cell lines.

표 3은 R-[ERBB2 압타머]-ODN-X (R = H, 콜레스테롤, 또는 PEG, X = H, 또는 idT)와 cODN-L-F18(L = linker)의 hybridization 구조를 R-[ERBB2 압타머]-X-hy(bp)-L-F18로 나타내었다.Table 3 shows the hybridization structure of R- [ERBB2 aptamer] -ODN-X (R = H, cholesterol, or PEG, X = H, or idT) and cODN-LF 18 (L = linker). Tamer] -X- hy (bp) -LF 18 .

도 7은 [AP001-24]-hy(bp)-L-F18의 microPET 이미지 결과이다.7 is [AP001-24] - hy (bp) is a result of the microPET image -LF 18.

도 8는 [AP001-24]-idT-hy(bp)-L-F18의 microPET 이미지 결과이다.8 shows microPET images of [AP001-24] -i dT- hy (bp) -LF 18 .

도 9는 콜레스테릴-[AP001-24]-hy(bp)-L-F18의 microPET 이미지 결과이다.9 is a cholesteryl - [AP001-24] - hy (bp ) is a result of the microPET image -LF 18.

도 10은 콜레스테릴-[AP001-24]-idT-hy(bp)-L-F18의 microPET 이미지 결과이다.10 shows microPET images of cholesteryl- [AP001-24] -i dT- hy (bp) -LF 18 .

도 11는 PEGylated-[AP001-24]-hy(bp)-L-F18의 microPET 이미지 결과이다.Figure 11 is PEGylated- [AP001-24] - hy (bp ) is a result of the microPET image -LF 18.

도 12는 PEGylated-[AP001-24]-idT-hy(bp)-L-F18의 microPET 이미지 결과이다.12 shows microPET images of PEGylated- [AP001-24] -i dT- hy (bp) -LF 18 .

도 13은 KPL4 암을 품은 마우스에서 [AP001-24]-hy(bp)-L-F18, [AP001-24]-idT-hy(bp)-L-F18, 콜레스테릴-[AP001-24]-hy(bp)-L-F18, 콜레스테릴-[AP001-24]-idT-hy(bp)-L-F18 의 비교영상.FIG. 13 shows [AP001-24] -hy (bp) -LF 18 , [AP001-24] -idT-hy (bp) -LF 18 , cholesteryl- [AP001-24] -hy in mice bearing KPL4 cancer. Comparative image of (bp) -LF 18 , cholesteryl- [AP001-24] -idT-hy (bp) -LF 18 .

도 14는 인간 유방암 세포주에서 웨스턴 블롯에 의한 HER2 발현도 결정. 14 also determines HER2 expression by Western blot in human breast cancer cell lines.

도 15는 HER2 항체와 ERBB2 압타머(AP001-25)를 이용하여 유방암 세포주의 유동 세포 계속법 분석 (a) 점도표는 항체로부터의 BT474와 (HER2 양성 세포주), MDA-MB231(HER2 음성 세포주)에 대한 형광 신호를 나타낸다. ERBB2 압타머(AP001-25) (빨강색) 또는 대조군 염기서열 (파란색) (b) 항체와 ERBB2 압타머(AP001-25) 그리고 음성 대조군을 이용한 두 세포주에서 유동 세포 분석 그래프.FIG. 15 shows flow cytometry analysis of breast cancer cell lines using HER2 antibody and ERBB2 aptamer (AP001-25). (A) Viscosity table shows BT474 from antibody and (HER2 positive cell line), MDA-MB231 (HER2 negative cell line). Indicates a fluorescence signal. ERBB2 aptamer (AP001-25) (red) or control sequence (blue) (b) Flow cytometry graphs in two cell lines using antibodies, ERBB2 aptamer (AP001-25) and negative controls.

도 16은 HER2 양성 세포주에서의 선별된 ERBB2 압타머(AP001-25)의 공초점 현미경 영상. (a) BT474, HER2 양성 유방암 세포주에서 FITC 표지 압타머로 변환, (b) MDA-MB231 암세포주에서 같은 ERBB2 압타머(AP001-25)의 처리(표지 DAPI:파란색, FITC-ERbB2 압타머:녹색).16 is a confocal microscopy image of selected ERBB2 aptamers (AP001-25) in HER2-positive cell lines. (a) BT474, conversion to FITC-labeled aptamers in HER2-positive breast cancer cell lines, (b) treatment of the same ERBB2 aptamer (AP001-25) in MDA-MB231 cancer cell lines (labeled DAPI: blue, FITC-ERbB2 aptamer: green) .

도 17는 BT474 암을 품은 마우스에서 18F 표지 ERBB2 압타머{[AP001-25]-hy(bp)-L-F18} 나타내는 PET 생체내의 영상. Hy(bp)는 hybridization(base pairing)으로 ODN/cODN hybridization을 나타냄.Figure 17 in a mouse cancer embraces a BT474 18 F labeled ERBB2 aptamer {[AP001-25] - hy (bp ) -LF 18} in a PET image representing the biometric. Hy (bp) represents ODN / cODN hybridization by hybridization (base pairing).

도 18은 암이 있는 마우스에서 18F 표지 ERBB2 압타머{[AP001-25]-hy(bp)-L-F18}의 생체분포 연구. 자료는 조직의 그람 당 주입된 활성화를 백분율로 나타낸 것이다(%ID/g). Error bars, SD (N=4).Studies of the biodistribution - {hy (bp) 18 -LF [AP001-25]} Fig. 18 is 18 F labeled ERBB2 aptamer in a mouse with cancer. Data is expressed as a percentage of activation injected per gram of tissue (% ID / g). Error bars, SD (N = 4).

도 19은 HER2 양성 및 음성 암이 있는 마우스에서 18F 표지 ERBB2 압타머{[AP001-25]-hy(bp)-L-F18} 나타내는 PET 생체 내의 영상들. (a) HER2 과발현 BT474 암(왼쪽 겨드랑이)은 (b) HER2 음성 MDA-MB231 암(오른쪽 겨드랑이)과 비교할 때 좀 더 증가된 섭취를 나타낸다. (c) 분당 숫자(CPM)와 암 조직에 그람당 주입된 양(%ID/g)을 18F 표지 ERBB2 압타머로 계산하였다. The PET image in the living body representing - {hy (bp) 18 -LF [AP001-25]} Fig. 19 is in mice with normal HER2 positive and negative arm 18 F labeled ERBB2 aptamer. (a) HER2 overexpression BT474 cancer (left armpit) shows increased uptake when compared to (b) HER2 negative MDA-MB231 cancer (right armpit). (c) Numbers per minute (CPM) and amount injected per gram of cancer tissue (% ID / g) were calculated with an 18 F labeled ERBB2 aptamer.

도 20은 HER2에 대한 H&E 및 IHC 염색 결과를 나타낸다(원본 배율 400X).20 shows H & E and IHC staining results for HER2 (original magnification 400 ×).

본 발명에 사용된 유방암 관련 Her2 수용체에 특이적으로 결합하는 ERBB2 압타머는 5'-TCAGCCGCCAGCCAGTTC-[Core sequence]-GACCAGAGCACCACAGAG-3'의 염기서열을 가지며 Core Sequence에서 숫자 6이나 첨부하는 염기서열 목록에서 n은 NaptyldU를 나타낸다. The ERBB2 aptamer that specifically binds to the breast cancer related Her2 receptor used in the present invention has a base sequence of 5'-TCAGCCGCCAGCCAGTTC- [Core sequence] -GACCAGAGCACCACAGAG-3 'and the number 6 in the Core Sequence or n in the attached base sequence list. Represents NaptyldU.

Description(클론 번호)Description (clone number) Core sequenceCore sequence 1One 9-ER-N-A01_A059-ER-N-A01_A05 A6G66AGAG666GCC6GAG6GCC6CG6AAGGGCG6AACAA (SEQ ID NO: 1)A6G66AGAG666GCC6GAG6GCC6CG6AAGGGCG6AACAA (SEQ ID NO: 1) 22 9-ER-N-A02_B059-ER-N-A02_B05 6AC6GGGCCCG66AGCC6C6GGCGC6CC66CGC66G6GCC (SEQ ID NO: 2)6AC6GGGCCCG66AGCC6C6GGCGC6CC66CGC66G6GCC (SEQ ID NO: 2) 33 9-ER-N-A03_C059-ER-N-A03_C05 66A6CAACGCAC6GAGGGCG6CAGC66C66666AGG (SEQ ID NO: 3)66A6CAACGCAC6GAGGGCG6CAGC66C66666AGG (SEQ ID NO: 3) 44 9-ER-N-A04_D059-ER-N-A04_D05 A6G6AGAG666GCC6GAG6GCC6CGCAAGGGCG6AACAG (SEQ ID NO: 4)A6G6AGAG666GCC6GAG6GCC6CGCAAGGGCG6AACAG (SEQ ID NO: 4) 55 9-ER-N-A06_E059-ER-N-A06_E05 6CC6G6CCCGG666ACACAAG66AAGGCAGCCGC6GGA6A (SEQ ID NO: 5)6CC6G6CCCGG666ACACAAG66AAGGCAGCCGC6GGA6A (SEQ ID NO: 5) 66 9-ER-N-B02_F059-ER-N-B02_F05 G6C6GAACACCGAGA66AGC6GAACGAACGG6A6GGACG6 (SEQ ID NO: 6)G6C6GAACACCGAGA66AGC6GAACGAACGG6A6GGACG6 (SEQ ID NO: 6) 77 9-ER-N-B03_G059-ER-N-B03_G05 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7) 88 9-ER-N-B04_H059-ER-N-B04_H05 CGCGA66AGA6GAACGCACAA6ACCCG66C6GAG6AAAG6 (SEQ ID NO: 8)CGCGA66AGA6GAACGCACAA6ACCCG66C6GAG6AAAG6 (SEQ ID NO: 8) 99 9-ER-N-B08_A069-ER-N-B08_A06 G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9)G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9) 1010 9-ER-N-B09_B069-ER-N-B09_B06 G66AGAC6GAACGCAC6GAGGGCCGCAGCC6A6C6GAAGG (SEQ ID NO: 10)G66AGAC6GAACGCAC6GAGGGCCGCAGCC6A6C6GAAGG (SEQ ID NO: 10) 1111 9-ER-N-B12_C069-ER-N-B12_C06 A6G66AGAG666GCC6GAG6GCC6CGCAAGGGCG6AACAA (SEQ ID NO: 11)A6G66AGAG666GCC6GAG6GCC6CGCAAGGGCG6AACAA (SEQ ID NO: 11) 1212 9-ER-N-C02_D069-ER-N-C02_D06 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7) 1313 9-ER-N-C03_E069-ER-N-C03_E06 G6C6GAGCA6CGCG666AGCCGAACGC6CGG6GAGG6AGA6 (SEQ ID NO: 12)G6C6GAGCA6CGCG666AGCCGAACGC6CGG6GAGG6AGA6 (SEQ ID NO: 12) 1414 9-ER-N-C05_F069-ER-N-C05_F06 6CA6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 13)6CA6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 13) 1515 9-ER-N-C06_G069-ER-N-C06_G06 C6ACACGAA6CAAC6CCCC6CCGCA6AC6GAACA6CACAA (SEQ ID NO: 14)C6ACACGAA6CAAC6CCCC6CCGCA6AC6GAACA6CACAA (SEQ ID NO: 14) 1616 9-ER-N-C08_H06 9-ER-N-C08_H06 66AGCAAAA6GCCA6G6GCG6CC6G6CCCGG666ACAGC (SEQ ID NO: 15)66AGCAAAA6GCCA6G6GCG6CC6G6CCCGG666ACAGC (SEQ ID NO: 15) 1717 9-ER-N-C10_A079-ER-N-C10_A07 6GA6G6CCCCAAC6CAGC6G6GAA6C6A6GCCCCCGCCCA (SEQ ID NO: 16)6GA6G6CCCCAAC6CAGC6G6GAA6C6A6GCCCCCGCCCA (SEQ ID NO: 16) 1818 9-ER-N-D01_B07 9-ER-N-D01_B07 C6GAGCGG66AC6ACACCACCG6GAGACC66AG66ACAAA (SEQ ID NO: 17)C6GAGCGG66AC6ACACCACCG6GAGACC66AG66ACAAA (SEQ ID NO: 17) 1919 9-ER-N-D02_C07 9-ER-N-D02_C07 A66AGA6GAAAGCGCA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 18)A66AGA6GAAAGCGCA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 18) 2020 9-ER-N-D03_D079-ER-N-D03_D07 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7) 2121 9-ER-N-D04_E079-ER-N-D04_E07 666GGAG6G6C66ACGG66GGAG6AA6CGAGGA6GGA6GA (SEQ ID NO: 19)666GGAG6G6C66ACGG66GGAG6AA6CGAGGA6GGA6GA (SEQ ID NO: 19) 2222 9-ER-N-D05_F079-ER-N-D05_F07 CCG66ACC6ACC6CC6CGACCG6GGG6GCCC66AG6CCCA (SEQ ID NO: 20)CCG66ACC6ACC6CC6CGACCG6GGG6GCCC66AG6CCCA (SEQ ID NO: 20) 2323 9-ER-N-D06_G079-ER-N-D06_G07 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCAGA (SEQ ID NO: 21)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCAGA (SEQ ID NO: 21) 2424 9-ER-N-D07_H079-ER-N-D07_H07 CCG66ACC6ACC6CC6CGACCG6GGG6GCC666AG6CCCA (SEQ ID NO: 22)CCG66ACC6ACC6CC6CGACCG6GGG6GCC666AG6CCCA (SEQ ID NO: 22) 2525 9-ER-N-D09_A089-ER-N-D09_A08 A6G66AGAG666GCC6GAG6GCC6CGCAAGGGCG6AACAA (SEQ ID NO: 23)A6G66AGAG666GCC6GAG6GCC6CGCAAGGGCG6AACAA (SEQ ID NO: 23) 2626 9-ER-N-D11_B089-ER-N-D11_B08 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAG6 (SEQ ID NO: 24)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAG6 (SEQ ID NO: 24) 2727 9-ER-N-E02_C08 9-ER-N-E02_C08 CCG66ACC6ACC6CC6CGACCG6GGG6GCCC66AG6CCCA (SEQ ID NO: 20)CCG66ACC6ACC6CC6CGACCG6GGG6GCCC66AG6CCCA (SEQ ID NO: 20) 2828 9-ER-N-E04_D089-ER-N-E04_D08 A66AGA6GAAAGCACA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 25)A66AGA6GAAAGCACA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 25) 2929 9-ER-N-E09_E08 9-ER-N-E09_E08 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7) 3030 9-ER-N-E11_F089-ER-N-E11_F08 A6G66AGAG666GCC6GAG6GCG6CGCAAGGGCG6AACAG (SEQ ID NO: 26)A6G66AGAG666GCC6GAG6GCG6CGCAAGGGCG6AACAG (SEQ ID NO: 26) 3131 9-ER-N-E12_G089-ER-N-E12_G08 6GAGAAGGGC6G6GCC66AC6CAAAA666GGGA6C6GAA (SEQ ID NO: 27)6GAGAAGGGC6G6GCC66AC6CAAAA666GGGA6C6GAA (SEQ ID NO: 27) 3232 9-ER-N-F01_H08 9-ER-N-F01_H08 G66AGAC6GAACGCAC6GAGGGCCGCAGCC6A6C6GAAGG (SEQ ID NO: 10)G66AGAC6GAACGCAC6GAGGGCCGCAGCC6A6C6GAAGG (SEQ ID NO: 10) 3333 9-ER-N-F02_A099-ER-N-F02_A09 G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9)G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9) 3434 9-ER-N-F03_B09 9-ER-N-F03_B09 G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9)G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9) 3535 9-ER-N-F04_C099-ER-N-F04_C09 G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9)G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9) 3636 9-ER-N-F05_D099-ER-N-F05_D09 6CC6GG6A6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 28)6CC6GG6A6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 28) 3737 9-ER-N-F08_E099-ER-N-F08_E09 6AGA6C6C6GA66AGG6AGAACGCCC6AC6C6AACGGCAG (SEQ ID NO: 29)6AGA6C6C6GA66AGG6AGAACGCCC6AC6C6AACGGCAG (SEQ ID NO: 29) 3838 9-ER-N-F09_F099-ER-N-F09_F09 6GAGAAGGGC6G6GCC66AC6CAAAA666GGGGA6C6GAA (SEQ ID NO: 30)6GAGAAGGGC6G6GCC66AC6CAAAA666GGGGA6C6GAA (SEQ ID NO: 30) 3939 9-ER-N-F11_G099-ER-N-F11_G09 6GAGAAGGGC6G6GCC66AC6CAAAA666GGGGA6C6GAA (SEQ ID NO: 31)6GAGAAGGGC6G6GCC66AC6CAAAA666GGGGA6C6GAA (SEQ ID NO: 31) 4040 9-ER-N-G02_H099-ER-N-G02_H09 A66AGA6GAAAGCGCA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 18)A66AGA6GAAAGCGCA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 18) 4141 9-ER-N-G03_A109-ER-N-G03_A10 G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9)G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9) 4242 9-ER-N-G04_B109-ER-N-G04_B10 CG6CC66GG6GAG666GGG6C6GAGCAGGAGCACG6GAG6 (SEQ ID NO: 32)CG6CC66GG6GAG666GGG6C6GAGCAGGAGCACG6GAG6 (SEQ ID NO: 32) 4343 9-ER-N-G08_C109-ER-N-G08_C10 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7) 4444 9-ER-N-G09_D109-ER-N-G09_D10 G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9)G6C6GAACACCGAGA66AGCCGAACGAACGG6A6GGACG6 (SEQ ID NO: 9) 4545 9-ER-N-H01_E109-ER-N-H01_E10 A66AGA6GAAAGCACA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 33)A66AGA6GAAAGCACA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 33) 4646 9-ER-N-H02_F109-ER-N-H02_F10 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7) 4747 9-ER-N-H03_G109-ER-N-H03_G10 A66AGA6GAAAGCACA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 34)A66AGA6GAAAGCACA66CCAACAACAGA6AA6C6GAGGG (SEQ ID NO: 34) 4848 9-ER-N-H04_H109-ER-N-H04_H10 G66AGAC6GAACGCAC6GAGGGCCGCAGCC6A6C6GAAGG (SEQ ID NO: 10)G66AGAC6GAACGCAC6GAGGGCCGCAGCC6A6C6GAAGG (SEQ ID NO: 10) 4949 9-ER-N-H08_A119-ER-N-H08_A11 6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7)6CC6GGCA6G66CGA6GGAGGCC666GA66ACAGCCCAGA (SEQ ID NO: 7) 5050 9-ER-N-H09_B119-ER-N-H09_B11 A6G66AGAG6C6GCC6GAG6GCC6CGCAAGGGCG6AACAG (SEQ ID NO: 35)A6G66AGAG6C6GCC6GAG6GCC6CGCAAGGGCG6AACAG (SEQ ID NO: 35)

6 = NapdU [5-(N-Naphthylcarboxyamide)-[0070] 2'-deoxyuridine]본 발명에서는 방사성 동위원소, 예를 18F, 32P, 123I, 89Zr, 67Ga, 201Tl, 111In-111나 형광염료, 예를 들어 시아닌 형광염료인 Cy3, Cy5, Cy7 등으로 표지된 HER2 압타머를 이용하여 생체 내 영상화를 위해 활용하였다. 본 발명의 실시 예에서는 동위원소나 형광염료로 표지된 ERBB2 압타머를 가지고 생체 내의 분자적 영상을 위한 표적 특이성과 임상 적용가능성에 대한 평가를 진행하였다.6 = NapdU [5- (N-Naphthylcarboxyamide)-[0070] 2'-deoxyuridine] In the present invention, radioisotopes, such as 18 F, 32 P, 123 I, 89 Zr, 67 Ga, 201 Tl, 111 In- HER2 aptamer labeled with 111 or a fluorescent dye, for example Cy3, Cy5, Cy7, etc., was used for in vivo imaging. In an embodiment of the present invention, an ERBB2 aptamer labeled with an isotope or a fluorescent dye was used to evaluate target specificity and clinical applicability for molecular imaging in vivo.

인간 상피 성장인자 수용체2 (HER2) 에 대한 ERBB2 압타머를 18F-fluoride 동위원소로 표지하였다. HER2가 발현된 암세포주로 압타머가 들어가는 것 확인하기 위하여 대조군 압타머와 같이 유동세포계수법과 공초점 현미경을 통하여 비교하였다. 18F이 표지된 HER2 특이 ERBB2 압타머를 양전자 단층 촬영하여 BT474 또는 KPL4 세포가 이식된 마우스에 대한 생체 분자 영상을 각각 시간별로 얻었다.ERBB2 aptamers for human epidermal growth factor receptor 2 (HER2) were labeled with 18 F-fluoride isotopes. In order to confirm that aptamer enters the HER2 expressing cancer cell line, flow cytometry and confocal microscopy were compared with control aptamer. Positron tomography of HER2-specific ERBB2 aptamers labeled with 18 F was used to obtain biomolecular images of mice transplanted with BT474 or KPL4 cells over time, respectively.

이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.

세포배양Cell culture

HER2가 발현된 인간 유방암 세포주인 BT474, KPL4, N87, 및 SK-BR-3를 가지고 시험관 및 생체 내 시험을 위해 이용하였다. 그리고 대조군으로 다른 인간 유방암 세포주 MDA-MB231를 가지고 실험을 진행하였다. 모든 세포주는 ATCC로부터 구매하였고 10% FBS를 함유한 MEM 배지에 배양하여 유지하였다.HER2 expressed human breast cancer cell lines BT474, KPL4, N87, and SK-BR-3 were used for in vitro and in vivo testing. And the experiment was conducted with another human breast cancer cell line MDA-MB231 as a control. All cell lines were purchased from ATCC and maintained in culture in MEM medium containing 10% FBS.

세포 용해, 웨스턴블롯Cell Lysis, Western Blot

세포 내 단백질을 추출하기 위해 단백질 분해효소 방지제가 포함된 세포 용해액을 얼음 위에서 30분간 배양하였다. 이렇게 얻어진 세포 용해액을 4℃ 20분간 원심분리하여 정제하였다. 단백질 정량을 위하여 Bradford법을 이용하여 정량화하였고 각각의 샘플에서 30㎍의 단백질 추출물을 10% SDS-PAGE로 전기영동하여 분리하였다. 그리고 나서 니트로셀룰로오스 막에 옮겨 HER2 항체와 대조군인 beta-action 항체를 프로브로 이용하여 ECL로 x-ray film에 감광하였다.Cell lysates containing protease inhibitors were incubated for 30 minutes on ice to extract intracellular proteins. The cell lysate thus obtained was purified by centrifugation at 4 ° C. for 20 minutes. Protein quantification was performed using the Bradford method and 30 μg of protein extract from each sample was isolated by electrophoresis with 10% SDS-PAGE. Then, it was transferred to the nitrocellulose membrane and sensitized to the x-ray film by ECL using a HER2 antibody and a control beta-action antibody as a probe.

ERBB2 압타머 합성ERBB2 aptamer synthesis

Her2-(+) 표적 ERBB2 압타머의 염기서열은 아래 표 2에 나타내었다. The base sequences of the Her2-(+) target ERBB2 aptamers are shown in Table 2 below.

[표 2]TABLE 2

[규칙 제91조에 의한 정정 28.06.2018]　

Figure WO-DOC-FIGURE-51
[Correction under Rule 91 28.06.2018]
Figure WO-DOC-FIGURE-51

[규칙 제91조에 의한 정정 28.06.2018]　

Figure WO-DOC-FIGURE-52
[Correction under Rule 91 28.06.2018]
Figure WO-DOC-FIGURE-52

ERBB2 압타머인 AP001-24는 타겟에 대한 결합력(Kd)가 3.1nM이고 AP001-25는 0.9nM이다.AP001-24, the ERBB2 aptamer, has a binding force (Kd) of 3.1 nM to the target and AP001-25 has 0.9 nM.

여기서 6은 다음 구조식의 NapdU [5-(N-나프틸카르복시아미드)-2'-데옥시우리딘]이고 A = 2'-데옥시아데노신, G = 2'-데옥시구아노신, C = 2'-데옥시시티딘이다. Wherein 6 is NapdU [5- (N-naphthylcarboxyamide) -2'-deoxyuridine] of the formula: A = 2'-deoxyadenosine, G = 2'-deoxyguanosine, C = 2 '-Deoxycytidine.

Figure PCTKR2018004770-appb-I000003
Figure PCTKR2018004770-appb-I000003

압타머 혼성(압타머 hybridization)을 위해서 ERBB2 압타머{[AP001-24] 및 [AP001-25]}의 3'에 fully matching sequence인 ODN(5'-CAGCCACACCACCAG-3')를 포함한 합성을 진행하였다.For aptamer hybridization, synthesis including ODN (5'-CAGCCACACCACCAG-3 '), which is a fully matching sequence at 3' of ERBB2 aptamers {[AP001-24] and [AP001-25]}, was performed. .

[AP001-24]-ODN 합성[AP001-24] -ODN Synthesis

5'-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-3' {[AP001-24]-ODN}를 다음과 같이 합성하였다. 5 '-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A] -CAG CCA CAC CAC CAG-3' {[AP001-24] -ODN} was synthesized as follows.

압타머 합성과정은 phosphoramidite 커플링 반응을 이용한 고체상 합성 방법을 이용하여 합성했으며 합성 후 t-부틸아민:메탄올:물(1:1:2 v/v/v) 용액 70℃, 5시간동안 반응을 통하여 cleavage와 deprotection 과정을 거쳐서 온전한 압타머를 얻은 후 건조시겼다. 합성 압타머를 HPLC로 [C18 칼럼(Waters, Xbridge OST C18 10x50mm, 260nm] 분리후 ESI MS 질량분석기 (Qtrap2000, ABI)를 이용하여 분자량을 측정하였다. The aptamer synthesis process was synthesized using the solid phase synthesis method using phosphoramidite coupling reaction, and the reaction was carried out for 5 hours at 70 ° C. in t -butylamine: methanol: water (1: 1: 2 v / v / v) Through cleavage and deprotection process, the whole aptamer was obtained and dried. Synthetic aptamer was separated by HPLC [C18 column (Waters, Xbridge OST C18 10x50mm, 260nm)] and then molecular weight was measured using an ESI MS mass spectrometer (Qtrap2000, ABI).

상기 표 1의 11번째 압타머(SEQ ID NO: 11)가 AP001-24에 해당한다. The 11th aptamer (SEQ ID NO: 11) of Table 1 corresponds to AP001-24.

[AP001-25]-ODN 합성 : [AP001-25] -ODN Synthesis:

5'-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-3'{[AP001-25]-ODN}를 앞에서 기술한 {[AP001-24]-ODN}합성방법으로 합성하였다.5 '-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A] -CAG CCA CAC CAC CAG-3' {[AP001-25] -ODN} as previously described {[AP001-24] -ODN} It synthesize | combined by the synthesis method.

표 1의 12번째 압타머(SEQ ID NO: 7)가 AP001-25에 해당한다. The 12th aptamer (SEQ ID NO: 7) in Table 1 corresponds to AP001-25.

같은 방식으로 CAG-3'{표 1의 (SEQ ID NO: 1~35)의 각 압타머-ODN}를 앞에서 기술한 {[AP001-24]-ODN}합성방법으로 합성하였다.In the same manner, CAG-3 '{each aptamer-ODN} of (SEQ ID NO: 1 to 35) in Table 1 was synthesized by the {[AP001-24] -ODN} synthesis method described above.

[AP001-24]-ODN-idT 합성[AP001-24] -ODN-idT Synthesis

5'-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-idT-3'{[AP001-24]-ODN-idT}를 idT(invert dT) CPG(Glen, 20-0302-10)를 사용하여 앞에서 기술한 {[AP001-24]- ODN } 합성방법으로 합성하였다.  5 '-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A] -CAG CCA CAC CAC CAG-idT-3' {[AP001-24] -ODN-idT} idT (invert dT) CPG (Glen , 20-0302-10) was synthesized by the {[AP001-24]-ODN} synthesis method described above.

[AP001-25]-ODN-idT 합성 : [AP001-25] -ODN-idT Synthesis:

5'-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-idT-3'{[AP001-25]-ODN-idT}를 idT CPG(Glen, 20-0302-10)를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다. 5 '-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A] -CAG CCA CAC CAC CAG-idT-3' {[AP001-25] -ODN-idT} idT CPG (Glen, 20-0302 -10) was synthesized using the {[AP001-24] -ODN} synthesis method described above.

콜레스테릴-[AP001-24]-ODN 합성Cholesteryl- [AP001-24] -ODN Synthesis

5'-콜레스테릴-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-3'{콜레스테릴-[AP001-24]-ODN}를 콜레스테롤-PA(Glen, 10-1976-90) 를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-cholesteryl- [6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A] -CAG CCA CAC CAC CAG-3 '{cholesteryl- [AP001-24] -ODN} to cholesterol-PA ( Glen, 10-1976-90) was used to synthesize the {[AP001-24] -ODN} synthesis method described above.

콜레스테릴-[AP001-25]-ODN 합성 : Cholesteryl- [AP001-25] -ODN Synthesis:

5'-콜레스테릴-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-3'{콜레스테릴-[AP001-25]-ODN}를 콜레스테롤-PA(Glen, 10-1976-90) 를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-cholesteryl- [A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A] -CAG CCA CAC CAC CAG-3 '{cholesteryl- [AP001-25] -ODN} to cholesterol-PA ( Glen, 10-1976-90) was used to synthesize the {[AP001-24] -ODN} synthesis method described above.

콜레스테릴-[AP001-24]-ODN-idT 합성Cholesteryl- [AP001-24] -ODN-idT Synthesis

5'-콜레스테릴-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-idT-3'{콜레스테릴-[AP001-24]-ODN-idT}를 idT CPG(Glen, 20-0302-10) 및 콜레스테롤-PA(Glen, 10-1976-90)를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-cholesteryl- [6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A] -CAG CCA CAC CAC CAG-idT-3 '{cholesteryl- [AP001-24] -ODN-idT} idT CPG (Glen, 20-0302-10) and cholesterol-PA (Glen, 10-1976-90) were used to synthesize the {[AP001-24] -ODN} synthesis method described above.

콜레스테릴-[AP001-25]-ODN-idT 합성 : Synthesis of cholesteryl- [AP001-25] -ODN-idT:

5'-콜레스테릴-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-idT-3'{콜레스테릴-[AP001-25]-ODN-idT}를 idT CPG(Glen, 20-0302-10) 및 콜레스테롤-PA(Glen, 10-1976-90)를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-cholesteryl- [A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A] -CAG CCA CAC CAC CAG-idT-3 '{cholesteryl- [AP001-25] -ODN-idT} idT CPG (Glen, 20-0302-10) and cholesterol-PA (Glen, 10-1976-90) were used to synthesize the {[AP001-24] -ODN} synthesis method described above.

PEGylated-[AP001-24]-ODN 합성PEGylated- [AP001-24] -ODN Synthesis

5'-PEGylated-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-3'{PEGylated-[AP001-24]-ODN}를 Polyethyleneglycol 2000 CED PA(ChemGenes, CLP-2119) 를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-PEGylated- [6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A] -CAG CCA CAC CAC CAG-3 '{PEGylated- [AP001-24] -ODN} Polyethyleneglycol 2000 CED PA (ChemGenes, CLP -2119) was synthesized using the {[AP001-24] -ODN} synthesis method described above.

PEGylated-[AP001-25]-ODN 합성 : PEGylated- [AP001-25] -ODN Synthesis:

5'-PEGylated-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-3'{PEGylated-[AP001-25]-ODN}를 Polyethyleneglycol 2000 CED PA(ChemGenes, CLP-2119) 를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-PEGylated- [A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A] -CAG CCA CAC CAC CAG-3 '{PEGylated- [AP001-25] -ODN} Polyethyleneglycol 2000 CED PA (ChemGenes, CLP -2119) was synthesized using the {[AP001-24] -ODN} synthesis method described above.

PEGylated-[AP001-24]-ODN-idT 합성PEGylated- [AP001-24] -ODN-idT Synthesis

5'-PEGylated-[6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A]-CAG CCA CAC CAC CAG-idT-3'{PEGylated-[AP001-24]-ODN-idT}를 idT CPG(Glen, 20-0302-10) 및 Polyethyleneglycol 2000 CED PA(ChemGenes, CLP-2119) 를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-PEGylated- [6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A] -CAG CCA CAC CAC CAG-idT-3 '{PEGylated- [AP001-24] -ODN-idT} idT CPG (Glen , 20-0302-10) and Polyethyleneglycol 2000 CED PA (ChemGenes, CLP-2119) were synthesized by the above-described synthesis method of {[AP001-24] -ODN}.

PEGylated-[AP001-25]-ODN-idT 합성 : PEGylated- [AP001-25] -ODN-idT Synthesis:

5'-PEGylated-[A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A]-CAG CCA CAC CAC CAG-3'{PEGylated-[AP001-25]-ODN-idT}를 idT CPG(Glen, 20-0302-10) 및 Polyethyleneglycol 2000 CED PA(ChemGenes, CLP-2119) 를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다.5'-PEGylated- [A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A] -CAG CCA CAC CAC CAG-3 '{PEGylated- [AP001-25] -ODN-idT} idT CPG (Glen, 20 -0302-10) and Polyethyleneglycol 2000 CED PA (ChemGenes, CLP-2119) were synthesized by the above-described {[AP001-24] -ODN} synthesis method.

Cy5 conjugated된 cODN(Complementary Oligonucleotide) [cODN-Cy5] 합성 :Synthesis of Cy5 conjugated cODN (Complementary Oligonucleotide) [cODN-Cy5]:

아래 그림은 cODN-Cy5 및 cODN-L-F18 (L = linker)구조 및 합성을 나타낸다.The figure below shows the structure and synthesis of cODN-Cy5 and cODN-LF 18 (L = linker).

Figure PCTKR2018004770-appb-I000004
Figure PCTKR2018004770-appb-I000004

5'-Cy5-[CTGGTGGTGTGGCTG]-3'[cODN-Cy5]를 Cy5-PA(Glen,10-5915-10)를 사용하여 앞에서 기술한 {[AP001-24]-ODN} 합성방법으로 합성하였다. 5'-Cy5- [CTGGTGGTGTGGCTG] -3 '[ cODN-Cy5 ] was synthesized using Cy5-PA (Glen, 10-5915-10) using the {[AP001-24] -ODN} synthesis method described above.

Cy5-표지 ERBB2 압타머 형성Cy5-labeled ERBB2 aptamer formation

아래 표 3에는 R-[ERBB2 압타머]-ODN-X (R = H, 콜레스테롤, 또는 PEG, X = H, 또는 idT)와 cODN-Cy5의 hybridization 구조를 R-[ERBB2 압타머]-X-hy(bp)-Cy5로 나타내었다.Table 3 below shows the hybridization structure of R- [ERBB2 aptamer] -ODN-X (R = H, cholesterol, or PEG, X = H, or i dT) and cODN-Cy5, R- [ERBB2 aptamer] -X hy (bp) -Cy5.

[표 3]TABLE 3

Figure PCTKR2018004770-appb-I000005
Figure PCTKR2018004770-appb-I000005

Cy5-표지 ERBB2 압타머, {R-[ERBB2 압타머]-X-hy(bp)-Cy5}를 다음과 같은 방식으로 만들었다.Cy5-labeled ERBB2 aptamer, {R- [ERBB2 aptamer] -X-hy (bp) -Cy5}, was made in the following manner.

먼저, 같은 몰수의 cODN-Cy5와 [ERBB2 압타머]-ODN을 annealing buffer(PBS)에 넣는다. 이때 MgCl2 의 농도가 최종 10mM 이 되도록 맞춘다. 위 반응물을 95℃에서 5분간 두었다가 상온에서 천천히 식힌다. cODN-Cy5 와 [ERBB2 압타머]-ODN의 Hybridization efficiency 는 전기영동(Typhoon FLA7000 3% 아가로오스 겔 analysis) 및 HPLC를 이용하여 분석하였다.(XBridge OST analytical column (2.5㎛, 4.6 × 50mm, Waters, 254 nm.0.1M TEAA/아세토니트릴) 도 2에 R-[ERBB2 aptamer]-ODN-X (R = H, 콜레스테롤, 또는 PEG, X = OH, 또는 idT)와 cODN-Cy5의 hybridization 결과물인 R-[ERBB2 aptamer]-X-hy(bp)-Cy5를 Typhoon FLA7000 3% 아가로오스 겔로 분석한 결과를 나타내었다. First, the same number of moles of cODN-Cy5 and [ERBB2 aptamer] -ODN are put in annealing buffer (PBS). Where MgCl 2 Adjust the concentration to be 10mM final. The reaction was allowed to stand at 95 ° C. for 5 minutes and then cooled slowly to room temperature. Hybridization efficiency of cODN-Cy5 and [ERBB2 aptamer] -ODN was analyzed by electrophoresis (Typhoon FLA7000 3% agarose gel analysis) and HPLC. (XBridge OST analytical column (2.5㎛, 4.6 × 50mm, Waters) , 254 nm. 0.1 M TEAA / acetonitrile) FIG. 2 shows R- [ERBB2 aptamer] -ODN-X (R = H, cholesterol, or PEG, X = OH, or idT) as a result of hybridization of cODN-Cy5 -[ERBB2 aptamer] -X- hy (bp) -Cy5 was analyzed by Typhoon FLA7000 3% agarose gel.

형광물질 Cy5가 표지된 합성 올리고뉴클레오타이드(cODN-Cy5)와 [ERBB2 압타머]-ODN의 상보적 염기 쌍합을 확인하였다. 콜레스테릴-[AP001-24]-ODN-idT, 콜레스테릴-[AP001-24]-ODN와 cODN-Cy5을 1:1 비율로 섞어준 다음 55℃, 60℃, 65℃에서 서로 결합하도록 온도를 유지하였다. 결합을 확인하기 위하여 3% 아가로오스 겔로 전기 영동을 한 뒤 Cy5는 FLA5000을 이용하여 형광을 영상화하였고 전체적인 압타머는 EtBr로 염색하여 UV로 영상화하였다. 결과를 도 3에 나타내었다. 콜레스테릴-[AP001-24]-ODN-idT, 콜레스테릴-[AP001-24]-ODN, PEGylated-[AP001-25]-ODN-idT, PEGylated-[AP001-25]-ODN와의 cODN-Cy5의 상보적 염기 쌍합을 비교하기 위하여 1:1로 섞어주고 95도에서 5분간 열을 가하여 결합한 뒤 위와 같은 방법으로 확인하였다. 콜레스테릴-[AP001-24]-ODN-idT, 콜레스테릴-[AP001-24]-ODN, PEGylated-[AP001-25]-ODN-idT, PEGylated-[AP001-25]-ODN와 형광 표지 ODN(cODN-Cy5) 사이의 상보적 염기 쌍합을 95도 가열과 가열하지 않은 것의 확인하여 비교하였다. 비교 결과는 도 4에 나타내었다. Complementary base pairing of synthetic oligonucleotide (cODN-Cy5) and [ERBB2 aptamer] -ODN labeled with fluorescent substance Cy5 was confirmed. Mix cholesteryl- [AP001-24] -ODN-idT, cholesteryl- [AP001-24] -ODN and cODN-Cy5 in a 1: 1 ratio, and then bond them together at 55 ° C, 60 ° C and 65 ° C. The temperature was maintained. After electrophoresis with 3% agarose gel to confirm binding, Cy5 was imaged with fluorescence using FLA5000, and the overall aptamer was stained with EtBr and imaged with UV. The results are shown in FIG. CODN- with cholesteryl- [AP001-24] -ODN-idT, cholesteryl- [AP001-24] -ODN, PEGylated- [AP001-25] -ODN-idT, PEGylated- [AP001-25] -ODN In order to compare the complementary base pairs of Cy5, the mixture was mixed in a 1: 1 ratio, heated for 5 minutes at 95 ° C, and confirmed by the above method. Cholesteryl- [AP001-24] -ODN-idT, Cholesteryl- [AP001-24] -ODN, PEGylated- [AP001-25] -ODN-idT, PEGylated- [AP001-25] -ODN and Fluorescent Labeling Complementary base pairings between ODN (cODN-Cy5) were compared with confirmation of heating at 95 degrees and no heating. Comparative results are shown in FIG. 4.

F18 방사성 동위원소 표지 cODN (Complementary Oligonucleotide) [cODN-L-F18]F 18 radioisotope labeling cODN (Complementary Oligonucleotide) [cODN-LF 18 ]

18F-labeled cODN 합성은 이미 보고된 과정(참고문헌 24)을 중심으로 하였다. No-carrier-added 18F-fluoride ion을 합성기(Tracerlab FXFN, GE Healthcare, Milwaukee, WI, USA)를 이용하여 생성 후 메실레이트(mesylate)와 반응(100℃에서 10분) 후 18F-fluoro-PEG-azide (18F-FPA)를 HPLC를 이용하여 정제하였다. 5'-hexynyl complementary oligonucleotide(5'-hex-cODN 200mg)에 아세토니트릴(10 mL) 중의 1M N,N-디이소프로필에틸아민, 아세토니트릴(20 mL) 중의 100 mM 요도드화 구리(I)를 넣고 18F-FPA (750e1100 MBq)를 넣어 click chemistry 반응을 진행하였다.(70℃, 20분간) 합성된 18F-labeled cODN(cODN-L-F18)은 HPLC H(Xbridge OST C18 10Х50mm, 용리액 아세토니트릴/0.1M TEAA 5:95-95:5 20분에 걸쳐, 유속: 5 mL/min, UV (254 nm)를 이용하여 정제하였다. 18 F-labeled cODN synthesis centered on a previously reported procedure (Ref. 24). No-carrier-added 18F-fluoride ion was generated using a synthesizer (Tracerlab FXFN, GE Healthcare, Milwaukee, WI, USA), followed by reaction with mesylate (10 minutes at 100 ° C) and 18 F-fluoro-PEG -azide (18F-FPA) was purified using HPLC. To 5'-hexynyl complementary oligonucleotide (5'-hex-cODN 200 mg) was added 1 M N, N-diisopropylethylamine in acetonitrile (10 mL) and 100 mM copper iodide (I) in acetonitrile (20 mL). 18F-FPA (750e1100 MBq) was added to proceed the click chemistry reaction (70 ° C., 20 minutes). The synthesized 18 F-labeled cODN (cODN-LF 18 ) was purified by HPLC H (Xbridge OST C18 10Х50mm, eluent acetonitrile / 0.1 M TEAA 5: 95-95: 5 over 20 minutes, flow rate: 5 mL / min, purification using UV (254 nm).

F18 방사성 동위원소 표지 ERBB2 압타머 [R-[ERBB2 압타머]-X-hy(bp)-L-F18] 형성Formation of F 18 radioisotope label ERBB2 aptamer [R- [ERBB2 aptamer] -X-hy (bp) -LF 18 ]

아래 표 4에 R-[ERBB2 aptamer]-ODN-X (R = H, 콜레스테롤, 또는 PEG, X = OH, 또는 idT)와 cODN-L-F18(L = linker)의 hybridization 구조를 R-[ERBB2 aptamer]-X-hy(bp)-L-F18로 나타내었다.Table 4 below shows the hybridization structure of R- [ERBB2 aptamer] -ODN-X (R = H, cholesterol, or PEG, X = OH, or idT) and cODN-LF 18 (L = linker). ] -X- hy (bp) -LF 18 .

[표 4]TABLE 4

Figure PCTKR2018004770-appb-I000006
Figure PCTKR2018004770-appb-I000006

F18 방사성 동위원소표지 ERBB2 압타머, {R-[ERBB2 압타머]-X-hy(bp)-L-F18}를 다음과 같은 방식으로 만들었다.The F 18 radioisotope label ERBB2 aptamer, {R- [ERBB2 aptamer] -X-hy (bp) -LF 18 }, was made in the following manner.

먼저, 같은 몰수의 cODN-L-F18 와 [ERBB2 압타머]-ODN을 annealing buffer(PBS)에 넣는다. 이때 MgCl2 의 농도가 최종 10mM 이 되도록 맞춘다. 위 반응물을 95℃에서 5분간 두었다가 상온에서 천천히 식힌다. cODN-L-F18와 [ERBB2 압타머]-ODN의 Hybridization efficiency 는 HPLC를 이용하여 분석하였다(XBridge OST analytical column (2.5 ㎛, 4.6 × 50mm, Waters, 254 nm.0.1M TEAA/아세토니트릴). 98% 이상의 혼성화율로 결합하였다.First, add equal moles of cODN-LF 18 and [ERBB2 aptamer] -ODN to annealing buffer (PBS). At this time, adjust the concentration of MgCl 2 to the final 10mM. The reaction was allowed to stand at 95 ° C. for 5 minutes and then cooled slowly to room temperature. Hybridization efficiency of cODN-LF 18 and [ERBB2 aptamer] -ODN was analyzed using HPLC (XBridge OST analytical column (2.5 μm, 4.6 × 50 mm, Waters, 254 nm.0.1 M TEAA / acetonitrile). 98% Combined with the above hybridization rate.

공초점 현미경Confocal microscope

BT474, KPL4, N87, SK-BR-3, 및 MDA-MB231 세포주를 coverslip에 분주하여 밤새 배양하였다. 약 80%정도가 자랐을 때 주의 깊게 씻어주고 형광이 표지된 ERBB2 압타머 {R-[ERBb2 압타머]-hy(bp)-Cy5}를 250nM의 농도로 처리하여 배양하였다. 배양 후에 조심스럽게 씻어주고 DAPI를 함유한 배양액을 슬라이드에 장착하였다. LSM700 공초점 현미경으로 형광을 관찰하였다. 현미경 셋팅은 FITC 관찰에는 488nm 레이저로 여기(excitation), 방출(emission)은 BP490-555 그리고 Texas red에서는 639nm 레이저로 방출은 LP640 필터를 이용하였다.BT474, KPL4, N87, SK-BR-3, and MDA-MB231 cell lines were aliquoted onto the coverslip and incubated overnight. Giving carefully wash time approximately 80% grow up a fluorescence labeled ERBB2 aptamer {R- [ERBb2 aptamer] - hy (bp) -Cy5} were incubated treated at a concentration of 250nM. After incubation, the cells were washed carefully and a medium containing DAPI was mounted on the slide. Fluorescence was observed with an LSM700 confocal microscope. The microscopic setting was an excitation with a 488nm laser for FITC observation, BP490-555 for emission, and a 6640nm laser for Texas red with LP640 filter.

앞선 실험과 같은 방법으로 ERBB2 과발현 유방암 세포주인 KPL4, N87 와 SK-BR-3를 coverslip에 분주하여 밤새 배양하였다. 약 80%정도가 자랐을 때 주의 깊게 씻어주고 Cy5형광이 표지된 ODN을 상보 염기 쌍합을 이용하여 ERBb2 압타머에 결합한 시료를 처리하여 배양하였다. 배양 후에 조심스럽게 씻어주고 DAPI를 함유한 배양액을 슬라이드에 장착하였다. LSM700 공초점 현미경으로 형광을 관찰하였다. In the same manner as in the previous experiment, ERBB2 overexpressing breast cancer cell lines KPL4, N87 and SK-BR-3 were dispensed onto the coverslip and incubated overnight. When about 80% grew, the cells were washed carefully and incubated by processing a sample in which Cy5 fluorescence-labeled ODN was bound to ERBb2 aptamer using complementary base pairing. After incubation, the cells were washed carefully and a medium containing DAPI was mounted on the slide. Fluorescence was observed with an LSM700 confocal microscope.

결과는 도 5에 나타내었다. The results are shown in FIG.

유동세포계수법Flow Cytometry

ERBB2 압타머의 특이도를 유동세포 계수시스템(BD Biosciences)을 이용하여 형광 활성 세포 분리법으로 검증하였다. BT474, KPL4, N87, SK-BR-3, 또는 MDA-MB231 암 세포주를 페트리디쉬에 적정 수를 계대 배양하여 80% 정도까지 자라도록 배양하였다. 세포에 트립신을 처리하고 PBS로 씻어준 다음 형광이 표지된 ODN을 온도에 의한 상보적 염기로 ERBB2 압타머에 결합하였다. 결합이 완료된 시료를 세포에 처리하였다. ERBB2 압타머 {R-[ERBb2 압타머]-hy(bp)-Cy5}와 대조군으로 1%FBS를 함유한 항체를 각각 4℃에서 30분간 처리하였다. 처리가 완료된 시료를 씻어준 다음 결합된 ERBB2 압타머를 측정하고 형광활성 세포 분리법을 통해 분석하였다.The specificity of ERBB2 aptamer was verified by fluorescence activated cell separation using flow cytometry (BD Biosciences). BT474, KPL4, N87, SK-BR-3, or MDA-MB231 cancer cell lines were cultured to pass up to 80% by passage of appropriate numbers in Petri dishes. The cells were trypsinized and washed with PBS, and the fluorescently labeled ODN was bound to ERBB2 aptamer as a complementary base by temperature. The completed sample was treated with the cells. ERBB2 aptamer {R- [ERBb2 aptamer] -hy (bp) -Cy5} and an antibody containing 1% FBS as a control were each treated at 4 ° C. for 30 minutes. The treated samples were washed and then bound ERBB2 aptamer was measured and analyzed by fluorescence-activated cell separation.

결과는 도 6에 나타내었다. The results are shown in FIG.

생체 내 실험In vivo experiment

4주령 Balb/c 누드 마우스 피하에 17ββ-estradiol 펠렛을 목 측면 부위에 에스트로겐이 암이 발생 가능할 정도의 양이 방출하도록 이식하였다. 몇일 후 마우스당 7X106 정도의 수로 BT474 또는 KPL4 인간 유방암 세포주를 피하에 이식하였다. 암이 3주간 발생되도록 한 후 암의 성장을 caliper로 측정하였다.17ββ-estradiol pellets were implanted subcutaneously in 4 week old Balb / c nude mice to release estrogens in cancerous areas. After a few days 7X10 6 per mouse BT474 or KPL4 human breast cancer cell lines were implanted subcutaneously in large numbers. After three weeks of cancer, the growth of the cancer was measured by a caliper.

Balb/C 누드 마우스에 인간 유방암 세포주인 KPL4세포를 1X105개를 오른쪽 어깨에 피하 이식하였다. 이후 암이 발생되도록 유도하였다.Balb / C nude mice were implanted subcutaneously with 1 × 10 5 human breast cancer cell line KPL4 cells in the right shoulder. It was then induced to develop cancer.

F18 방사성 동위원소 표지 ERBB2 압타머 PET 영상화F 18 Radioisotope Labeling ERBB2 Aptamer PET Imaging

마우스에 F18 방사성 동위원소 표지 ERBB2 압타머 주사 후 60분 후부터 10분간의 정적 영상을 Inveon microPET (Siemens, Knoxville, TN, USA) 스캐너를 이용하여 획득하였다. F18 방사성 동위원소 표지 ERBB2 압타머 주사 시 2% Isoflurane으로 호흡 마취한 후 마우스의 꼬리 정맥에 7.4 MBq의 F18 방사성 동위원소 표지 ERBB2 압타머를 주사하였다. 획득된 리스트모드 (listmode) 데이터는 사이노그램으로 변환 후 3D Ordered Subset Expectation Maximization (OSEM) 알고리즘으로 재구성하여 ASIpro (Concorde Microsystems Inc, Knoxville, TN)를 이용하여 분석하였다.Mice were acquired using Inveon microPET (Siemens, Knoxville, TN, USA) scanner for 60 minutes from 60 minutes after F 18 radioisotope labeled ERBB2 aptamer injection. Respiratory anesthesia with 2% Isoflurane upon F 18 radioisotope labeled ERBB2 aptamer injection followed by injection of 7.4 MBq of F 18 radioisotope labeled ERBB2 aptamer into the tail vein of mice. The obtained listmode data was transformed into sinograms and reconstructed with a 3D Ordered Subset Expectation Maximization (OSEM) algorithm and analyzed using ASIpro (Concorde Microsystems Inc, Knoxville, TN).

F18 방사성 동위원소 표지 ERBB2 압타머를 인간 유방암 세포주입으로 종양을 키운 마우스에 정맥 주사한 후에 지멘스사의 inveopn PET를 이용하여 PET를 수행하였다. 주입한 양은 13.7 ± 1.1 MBq (370 ± 30 uCi)였고 역학 PET 연구는 10개 1분 영상과 4개 5분 영상 프로토콜에 따라 30분 동안 수행되었다. 두 정지 상태에서의 연구가 주입 후 10, 60, 90 그리고 120분간 수행되었다. PET 신호의 부분정량은 AMIDE 소프트웨어를 이용하여 수행되었다. 영상들은 양전자 표지 프로브의 조직 농도(%ID/g)에 비례하는 false-color scale를 이용하여 구현하였다. 빨강색은 가장 농도가 높은 것을 나타내어주고 노랑, 녹색, 파랑은 점차적으로 낮은 농도와 일치한다. F 18 radioactive isotopic label ERBB2 The aptamer was injected intravenously into tumor-grown mice by human breast cancer cell injection, and then PET was performed using Siemens inveopn PET. The amount injected was 13.7 ± 1.1 MBq (370 ± 30 uCi) and the dynamic PET study was performed for 30 minutes according to 10 1 minute images and 4 5 minute imaging protocols. Two stationary studies were performed for 10, 60, 90 and 120 minutes after infusion. Partial quantification of PET signals was performed using AMIDE software. Images were implemented using a false-color scale proportional to the tissue concentration (% ID / g) of the positron labeled probe. Red indicates the highest concentration, and yellow, green, and blue gradually match the lower concentrations.

PET 이미지를 도 7 내지 도 13에 나타내었다. PET images are shown in FIGS. 7-13.

결과result

HER2 발현의 입증과 표적 암세포에 대한 압타머의 친화도Demonstration of HER2 Expression and Affinity of Aptamers to Target Cancer Cells

웨스턴블럿과 유동 세포 계수법이 유방암 세포주인 BT474의 발현을 조사하기 위해 수행되었다. 웨스턴블롯 분석은 유전자 증폭에 기인하여 HER2가 과발현한다고 알려진 SKBR3세포주 뿐만 아니라 BT474에서의 과발현을 확인하였다. 음성 대조군 세포주인 MDA-MB231에서는 해당하는 위치에서 신호가 보이지 않는 것을 확인하였다 (도 14).Western blot and flow cytometry were performed to investigate expression of breast cancer cell line BT474. Western blot analysis confirmed overexpression in BT474 as well as SKBR3 cell line, which is known to overexpress HER2 due to gene amplification. In the negative control cell line MDA-MB231, it was confirmed that no signal was seen at the corresponding position (FIG. 14).

도 15에서 보이는 바와 같이 HER2 항체는 유동 세포 계수법을 이용하여 HER2-양성 BT474세포주에 매우 특이적으로 결합하는 것으로 보여진다. 항체와 비교할 떄 ERBB2 압타머는 MDA-MB231 세포주에서는 매우 약한 반면 BT474 세포주에서는 강하게 결합하는 것을 볼 수 있다. 게다가 무작위 핵산 올리고에서는 어떠한 세포주에서도 결합하는 것을 볼 수 없었다. 이러한 결과는 ERBB2 압타머가 HER2 양성 유방암 세포주에 우선적으로 결합한다는 것을 제시하고 이는 이러한 세포주 표면에 HER2 구조를 인지함으로 인하여 가능하다. 같은 방법으로 유방암 세포주인 KPL4와 SK-BR-3 세포주 형광 표지 압타머가 강하게 결합하는 것이 관찰되었다.As shown in FIG. 15, the HER2 antibody was shown to bind very specifically to the HER2-positive BT474 cell line using flow cytometry. Compared with antibodies, it can be seen that ERBB2 aptamers are very weak in the MDA-MB231 cell line but strongly bind in the BT474 cell line. In addition, no binding to any cell line was seen in random nucleic acid oligos. These results suggest that ERBB2 aptamers preferentially bind to HER2-positive breast cancer cell lines, which is possible by recognizing the HER2 structure on the surface of these cell lines. In the same manner, strong binding of the breast cancer cell line KPL4 and the SK-BR-3 cell line fluorescent label aptamer was observed.

공초점 현미경분석Confocal Microscopy

ERBB2 압타머의 세포 결합은 공초점 현미경으로 좀 더 평가되었다(도 16). BT474 HER2 양성 유방암 세포주에 압타머를 처리하였다. ERbB2 압타머는 형광으로 표지가 되어 세포 표면에서 형광이 관측이 되었고 이러한 세포의 표면에 HER2 구조체가 있는 것으로 확인되었다. 세포막을 따라 압타머가 나타내는 형광이 관측되었고 음성 대조군인 MDA-MB231 세포주에서는 아무런 형광신호도 관측되지 않았으므로 HER2가 존재하지 않는 것으로 보인다. 따라서 이 ERBB2 압타머는 HER2 양성 유방암 세포주에 결합할 수 있고 HER2 음성 세포에는 최소 결합하는 것으로 관찰되었다. 형광표지 ODN과 상보적 염기 쌍합을 이룬 ERBB2 압타머{[AP001-24] 및 [AP001-25번]}을 위에서 진행한 실험과 같은 방법으로 유방암 세포주인 KPL4, N87, SK-BR-3에 처리한 뒤 공초점 현미경으로 형광을 관찰하였다. 두 종의 ERBB2 압타머 모두 유방암 세포주에 잘 결합하는 것이 확인되었고 [AP001-24]는 세포막을 따라 세포 표면에 형광이 관측되었고 [AP001-25]는 세포 내부에서도 형광이 관측되었다.Cell binding of ERBB2 aptamer was further evaluated by confocal microscopy (FIG. 16). BT474 HER2-positive breast cancer cell lines were treated with aptamers. The ERbB2 aptamer was labeled with fluorescence and fluorescence was observed on the cell surface, and it was confirmed that the HER2 structure was present on the surface of these cells. The fluorescence represented by the aptamer was observed along the cell membrane, and no fluorescence signal was observed in the negative control MDA-MB231 cell line. Thus, it was observed that this ERBB2 aptamer can bind to HER2-positive breast cancer cell lines and minimal binding to HER2 negative cells. Treatment of ERBB2 aptamers {[AP001-24] and [AP001-25]} with complementary base pairs with fluorescently labeled ODN was performed on KPL4, N87 and SK-BR-3 breast cancer cell lines in the same manner as the experiments conducted above. After fluorescence was observed under a confocal microscope. Both ERBB2 aptamers were found to bind well to breast cancer cell lines, [AP001-24] was observed at the cell surface along the cell membrane, and [AP001-25] was also observed inside the cell.

생체 내 PET 영상, 생체분포, 면역조직화학In vivo PET imaging, biodistribution, immunohistochemistry

동물 micro PET을 이용하여 BT474 또는 KPL4암을 품은 마우스의 생체 내 분자 이미지가 시간별로 주어졌다. 도 17에 따르면 18F 표지 HER2 특이 ERBB2 압타머가 마우스의 왼쪽 겨드랑이에 존재하는 암 조직에 섭취가 매우 증가하는 것이 관찰되었다. 120분간 주어진 영상에서 암은 수평면과 관상면 영상에서 ERBB2 압타머에 의해 분명하게 표지되었다. 창자와 방광에서의 생리적인 섭취가 뚜렷하게 나타난 것으로 보아 방사선의약품의 두 가지 주요 배출 경로를 반영한다.In vivo molecular images of mice bearing BT474 or KPL4 cancers were given over time using animal micro PET. According to FIG. 17, it was observed that the intake of 18F-labeled HER2-specific ERBB2 aptamer was significantly increased in cancer tissues present in the left armpit of the mouse. In the 120 min given image, the cancer was clearly marked by the ERBB2 aptamer in horizontal and coronal images. The apparent physiological intake of the intestines and bladder reflects two major routes of release of radiopharmaceuticals.

생체분포는 18F로 표지된 ERBB2 압타머의 주입 후 한 시간 후에 암을 품은 마우스에서 검증되었다. 동물을 희생시킨 후에 암을 포함한 각각의 조직에서의 방사능 수치를 감마 계측기를 이용하여 측정하여 %ID/g를 표현하였다(도 18). 아래 표 5 에도 나타내었다. Biodistribution was validated in mice bearing cancer one hour after injection of ERBB2 aptamer labeled with 18 F. After sacrificing the animals, radioactivity levels in each tissue, including cancer, were measured using a gamma meter to express% ID / g (FIG. 18). It is also shown in Table 5 below.

[표 5]TABLE 5

Figure PCTKR2018004770-appb-I000007
Figure PCTKR2018004770-appb-I000007

18F 표지 ERBB2 압타머의 암에서 섭취는 한 시간당 0.62±±0.04였다. 생체 분포도에 대한 연구는 18F 표지 ERBB2 압타머 두 가지 주요 배출 경로가 신장과 창자임을 보여준다. The intake of 18 F labeled ERBB2 aptamers was 0.62 ± 0.04 per hour. Studies on biodistribution show that the two major release pathways of the 18 F-labeled ERBB2 aptamer are the kidneys and intestines.

도 19는 HER2 양성과 음성 암을 품은 마우스에서 18F 표지 ERBb2 압타머를 나타내는 영상을 보여준다. HER2 과발현 BT474암은 HER2 음성 MSA-MB231 암과 비교할때 높은 동위원소 섭취를 보여준다. 반정량적 분석을 위해 각각 VOI(voxel- 또는 volume-of-interest)중의 전체 활성(nCi)이 계산되었다. BT474와 MDA-MB231 세포주 사이의 T/M (tumor/muscle) 섭취 비율의 비교는 BT474 암에 과발현 HER2에서 T/M 비율과 대조 이미지가 높은 것으로 보인다(도 19). 면역조직화학으로 각각의 마우스 그룹으로부터 적출된 BT474암에서 HER2의 높은 발현과 MDA-MB231 세포에서의 HER2가 낮게 발현되는 것이 확인되었다(도 20). BT474 암세포(윗줄)는 MDA-MB231 세포주(아랫줄)와 비교할 때 HER2에 대해 더 강하게 세포막이 염색되는 것이 관찰된다.19 shows images showing 18 F labeled ERBb2 aptamers in mice bearing HER2 positive and negative cancers. HER2 overexpression BT474 cancer shows high isotope uptake compared to HER2 negative MSA-MB231 cancer. For semiquantitative analysis, the total activity (nCi) in VOI (voxel- or volume-of-interest), respectively, was calculated. Comparison of the T / M (tumor / muscle) uptake rate between BT474 and MDA-MB231 cell lines appears to have a high T / M ratio and contrast image in overexpressed HER2 in BT474 cancer (FIG. 19). Immunohistochemistry confirmed high expression of HER2 and low expression of HER2 in MDA-MB231 cells in BT474 cancer isolated from each mouse group (FIG. 20). BT474 cancer cells (upper row) are observed to stain the cell membrane more strongly against HER2 compared to the MDA-MB231 cell line (lower row).

본 발명에서, HER2 표적 ERBB2 압타머를 생체 내에서 성공적으로 PET 영상화하였다. 본 발명은 특이적인 ERBB2 압타머를 이용하여 HER2 표적 PET영상화한 첫 번째 사례이다. BT474 암이 있는 마우스에서 PET 영상들은 ERBB2 압타머가 생체 내에서 HER2를 인지하여 암을 비교적 선명하게 보여주는 것으로 확인되었다. 이러한 결과는 방사선 표지 ERBB2 압타머가 HER2 양성 유방암 세포주에 대한 표적 치료에 응용되거나 치료방법을 결정하는데 잠재적으로 적용될 수 있다.In the present invention, HER2 target ERBB2 aptamer was successfully PET imaged in vivo. The present invention is the first case of HER2 target PET imaging using specific ERBB2 aptamer. PET images from mice with BT474 cancer were confirmed by the ERBB2 aptamer to recognize HER2 in vivo and show the cancer relatively clearly. These results could potentially be applied to the radiotherapy-labeled ERBB2 aptamer or to determine the treatment method for targeted treatment of HER2-positive breast cancer cell lines.

상기 실시 예에서 확인한 바와 같이, R-[ERBB2 aptamer]-ODN-X 와 cODN-L-F18을 결합시켜 R-[ERBB2 aptamer]-ODN-X/cODN-L-F18[상기에서 R-[ERBB2 aptamer]-X-hy(bp)-L-F18로 표시]을 제조했을 때, R = H( No Protecting), X = H (No protecting)에서 R = 콜레스테롤, PEG(폴리에틸렌글리콜), X = idT(inverted deoxythymidine), LNA(Locked Nucleic Acid), 2’-메톡시 뉴클레오타이드, 2’-아미노 뉴클레오타이드, 2’F-뉴클레오타이드 등으로 5’ 말단 위치나 3’ 말단 위치, 또는 양쪽 모두에서 화학적으로 변형된(protecting) 압타머인 경우 영상 이미지가 좋아진다. As confirmed in the above embodiment, R- [ERBB2 aptamer] -ODN-X and cODN-L-F18 were combined to R- [ERBB2 aptamer] -ODN-X / cODN-L-F18 [R- [ERBB2 aptamer] -X-hy (bp) -L-F18], where R = H (No Protecting), X = H (No protecting), R = cholesterol, PEG (polyethylene glycol), X = idT (inverted deoxythymidine), LNA (Locked Nucleic Acid), 2'-methoxy nucleotides, 2'-amino nucleotides, 2'F-nucleotides, etc. chemically modified at the 5 'end position or 3' end position, or both (protecting) Aptamers get better image quality.

이들 화합물에 의한 변형에 의해 t1/2 blood clearence를 높여주는 효과. 즉, 체내에서 혈중 내 반감기를 높여줌으로써 방사성 동위원소를 결합시킨 ERBB2 압타머가 종양에 더 많이 결합하여 영상효율을 높여주는 효과를 나타낸다[R = H, X = H 가 t1/2 = 10min인데 비하여 R과 X가 보호되어 변형된 경우 1시간 증가함으로써 영상 이미지가 좋아짐을 확인하였다.Modification by these compounds increases the t1 / 2 blood clearence. In other words, by increasing the half-life in the blood, the radioactive isotope bound ERBB2 aptamer binds more to the tumor, thereby improving the image efficiency [R = H, X = H is t1 / 2 = 10min compared to R When X and X were protected and deformed, the image was improved by 1 hour.

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<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-A04_D05, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-A04_D05, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 4<400> 4

angnagagnnngccngagngccncgcaagggcgnaacag 39angnagagnnngccngagngccncgcaagggcgnaacag 39

<210> 5<210> 5

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-A06_E05, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-A06_E05, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 5<400> 5

nccngncccggnnnacacaagnnaaggcagccgcnggana 40nccngncccggnnnacacaagnnaaggcagccgcnggana 40

<210> 6<210> 6

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-B02_F05, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-B02_F05, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 6<400> 6

gncngaacaccgagannagcngaacgaacggnanggacgn 40gncngaacaccgagannagcngaacgaacggnanggacgn 40

<210> 7<210> 7

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-B03_G05, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-B03_G05, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 7<400> 7

nccnggcangnncganggaggccnnngannacagcccaga 40nccnggcangnncganggaggccnnngannacagcccaga 40

<210> 8<210> 8

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-B04_H05, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-B04_H05, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 8<400> 8

cgcgannagangaacgcacaanacccgnncngagnaaagn 40cgcgannagangaacgcacaanacccgnncngagnaaagn 40

<210> 9<210> 9

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-B08_A06, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-B08_A06, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 9<400> 9

gncngaacaccgagannagccgaacgaacggnanggacgn 40gncngaacaccgagannagccgaacgaacggnanggacgn 40

<210> 10<210> 10

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-B09_B06, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-B09_B06, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 10<400> 10

gnnagacngaacgcacngagggccgcagccnancngaagg 40gnnagacngaacgcacngagggccgcagccnancngaagg 40

<210> 11<210> 11

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-B12_C06, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-B12_C06, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 11<400> 11

angnnagagnnngccngagngccncgcaagggcgnaacaa 40angnnagagnnngccngagngccncgcaagggcgnaacaa 40

<210> 12<210> 12

<211> 41<211> 41

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-C03_E06, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-C03_E06, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 12<400> 12

gncngagcancgcgnnnagccgaacgcncggngaggnagan 41gncngagcancgcgnnnagccgaacgcncggngaggnagan 41

<210> 13<210> 13

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-C05_F06, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-C05_F06, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 13<400> 13

ncanggcangnncganggaggccnnngannacagcccaga 40ncanggcangnncganggaggccnnngannacagcccaga 40

<210> 14<210> 14

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-C06_G06, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-C06_G06, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 14<400> 14

cnacacgaancaacnccccnccgcanacngaacancacaa 40cnacacgaancaacnccccnccgcanacngaacancacaa 40

<210> 15<210> 15

<211> 39<211> 39

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-C08_H06, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-C08_H06, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 15<400> 15

nnagcaaaangccangngcgnccngncccggnnnacagc 39nnagcaaaangccangngcgnccngncccggnnnacagc 39

<210> 16<210> 16

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-C10_A07, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-C10_A07, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 16<400> 16

ngangnccccaacncagcngngaancnangcccccgccca 40ngangnccccaacncagcngngaancnangcccccgccca 40

<210> 17<210> 17

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D01_B07, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D01_B07, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 17<400> 17

cngagcggnnacnacaccaccgngagaccnnagnnacaaa 40cngagcggnnacnacaccaccgngagaccnnagnnacaaa 40

<210> 18<210> 18

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D02_C07, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D02_C07, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 18<400> 18

annagangaaagcgcannccaacaacaganaancngaggg 40annagangaaagcgcannccaacaacaganaancngaggg 40

<210> 19<210> 19

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D04_E07, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D04_E07, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 19<400> 19

nnnggagngncnnacggnnggagnaancgaggangganga 40nnnggagngncnnacggnnggagnaancgaggangganga 40

<210> 20<210> 20

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D05_F07, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D05_F07, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 20<400> 20

ccgnnaccnaccnccncgaccgngggngcccnnagnccca 40ccgnnaccnaccnccncgaccgngggngcccnnagnccca 40

<210> 21<210> 21

<211> 39<211> 39

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D06_G07, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D06_G07, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 21<400> 21

nccnggcangnncganggaggccnnngannacagccaga 39nccnggcangnncganggaggccnnngannacagccaga 39

<210> 22<210> 22

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D07_H07, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D07_H07, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 22<400> 22

ccgnnaccnaccnccncgaccgngggngccnnnagnccca 40ccgnnaccnaccnccncgaccgngggngccnnnagnccca 40

<210> 23<210> 23

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D09_A08, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D09_A08, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 23<400> 23

angnnagagnnngccngagngccncgcaagggcgnaacaa 40angnnagagnnngccngagngccncgcaagggcgnaacaa 40

<210> 24<210> 24

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-D11_B08, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-D11_B08, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 24<400> 24

nccnggcangnncganggaggccnnngannacagcccagn 40nccnggcangnncganggaggccnnngannacagcccagn 40

<210> 25<210> 25

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-E04_D08, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-E04_D08, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 25<400> 25

annagangaaagcacannccaacaacaganaancngaggg 40annagangaaagcacannccaacaacaganaancngaggg 40

<210> 26<210> 26

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-E11_F08, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-E11_F08, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 26<400> 26

angnnagagnnngccngagngcgncgcaagggcgnaacag 40angnnagagnnngccngagngcgncgcaagggcgnaacag 40

<210> 27<210> 27

<211> 39<211> 39

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-E12_G08, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-E12_G08, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 27<400> 27

ngagaagggcngngccnnacncaaaannngggancngaa 39ngagaagggcngngccnnacncaaaannngggancngaa 39

<210> 28<210> 28

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-F05_D09, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-F05_D09, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 28<400> 28

nccnggnangnncganggaggccnnngannacagcccaga 40nccnggnangnncganggaggccnnngannacagcccaga 40

<210> 29<210> 29

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-F08_E09, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-F08_E09, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 29<400> 29

nagancncngannaggnagaacgcccnacncnaacggcag 40nagancncngannaggnagaacgcccnacncnaacggcag 40

<210> 30<210> 30

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-F09_F09, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-F09_F09, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 30<400> 30

ngagaagggcngngccnnacncaaaannnggggancngaa 40ngagaagggcngngccnnacncaaaannnggggancngaa 40

<210> 31<210> 31

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-F11_G09, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-F11_G09, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 31<400> 31

ngagaagggcngngccnnacncaaaannnggggancngaa 40ngagaagggcngngccnnacncaaaannnggggancngaa 40

<210> 32<210> 32

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-G04_B10, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-G04_B10, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 32<400> 32

cgnccnnggngagnnngggncngagcaggagcacgngagn 40cgnccnnggngagnnngggncngagcaggagcacgngagn 40

<210> 33<210> 33

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-H01_E10, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-H01_E10, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 33<400> 33

annagangaaagcacannccaacaacaganaancngaggg 40annagangaaagcacannccaacaacaganaancngaggg 40

<210> 34<210> 34

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-H03_G10, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-H03_G10, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 34<400> 34

annagangaaagcacannccaacaacaganaancngaggg 40annagangaaagcacannccaacaacaganaancngaggg 40

<210> 35<210> 35

<211> 40<211> 40

<212> DNA<212> DNA

<213> Artificial Sequence<213> Artificial Sequence

<220><220>

<223> core sequence represented by Clone No. 9-ER-N-H09_B11, wherein n is NapdU [5-(N-Naphthylcarboxyamide)-2'-deoxyuridine]<223> core sequence represented by Clone No. 9-ER-N-H09_B11, wherein n is NapdU [5- (N-Naphthylcarboxyamide) -2'-deoxyuridine]

<400> 35<400> 35

angnnagagncngccngagngccncgcaagggcgnaacag 40angnnagagncngccngagngccncgcaagggcgnaacag 40

17                                               17

Claims (10)

방사성 동위원소 또는 형광염료로 표지된, 염기서열 목록 번호(SEQ ID NO) 1~35에서 선택된 염기서열을 포함하는, HER2 특이성 ERBB2 압타머를 포함하는 종양성 질환 부위의 영상화용 조성물.Composition for imaging a tumor disease site comprising a HER2 specific ERBB2 aptamer, comprising a nucleotide sequence selected from SEQ ID NO: 1 to 35, labeled with a radioisotope or fluorescent dye. 제1항에 있어서, 상기 압타머는 다음의 서열번호 AP001-24 또는 서열번호 AP001-25의 염기서열을 포함하는 것을 특징으로 하는 영상화용 조성물:The composition for imaging according to claim 1, wherein the aptamer comprises a nucleotide sequence of the following SEQ ID NO: AP001-24 or SEQ ID NO: AP001-25: AP001-24: 5'-A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A-3' AP001-24: 5'-A6G 66A GAG 666 GCC 6GA G6G CC6 CGC AAG GGC G6A ACA A-3 ' AP001-25: 5'-6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A-3' AP001-25: 5'-6CC 6GG CA6 G66 CGA 6GG AGG CC6 66G A66 ACA GCC CAG A-3 ' 여기서 6은 NapdU [5-(N-나프틸카르복시아미드)-2’-데옥시우리딘]이고 A = 2’-데옥시아데노신, G = 2’-데옥시구아노신, C = 2’-데옥시시티딘이다.Wherein 6 is NapdU [5- (N-naphthylcarboxyamide) -2'-deoxyuridine] and A = 2'-deoxyadenosine, G = 2'-deoxyguanosine, C = 2'-de Oxycytidine. 제1항에 있어서, 상기 압타머는 5’ 말단 위치 및 3’ 말단 위치 중 어느 하나 이상의 위치에서 화학적으로 변형된 압타머인 것을 특징으로 하는 영상화용 조성물.The composition of claim 1, wherein the aptamer is a chemically modified aptamer at any one or more of a 5 ′ end position and a 3 ′ end position. 제3항에 있어서, 상기 변형은 콜레스테롤 또는 PEG(폴리에틸렌글리콜)에 의해 행해지는 것을 특징으로 하는 영상화용 조성물.The composition for imaging according to claim 3, wherein the modification is performed by cholesterol or polyethylene glycol (PEG). 제3항에 있어서, 상기 변형은 idT(inverted deoxythymidine), LNA(Locked Nucleic Acid), 2’-메톡시 뉴클레오타이드, 2’-아미노 뉴클레오타이드, 또는 2’F-뉴클레오타이드)에 의해 행해지는 것을 특징으로 하는 영상화용 조성물.The method of claim 3, wherein the modification is performed by inverted deoxythymidine (IDT), Locked Nucleic Acid (LNA), 2'-methoxy nucleotides, 2'-amino nucleotides, or 2'F-nucleotides). Imaging composition. 제1항에 있어서, 상기 동위원소는 18F, 32P, 123I, 89Zr, 67Ga, 201Tl, 111In-111에서 선택되는 것을 특징으로 하는 영상화용 조성물.The composition of claim 1, wherein the isotope is selected from 18 F, 32 P, 123 I, 89 Zr, 67 Ga, 201 Tl, 111 In-111. 제6항에 있어서, 상기 동위원소는 18F인 것을 특징으로 하는 영상화용 조성물.The composition of claim 6, wherein the isotope is 18 F. 8. 제1항에 있어서, 상기 형광염료는 시아닌 형광염료인 것을 특징으로 하는 영상화용 조성물.The composition for imaging according to claim 1, wherein the fluorescent dye is a cyanine fluorescent dye. 제8항에 있어서, 상기 형광염료는 Cy5인 것을 특징으로 하는 영상화용 조성물.The composition for imaging according to claim 8, wherein the fluorescent dye is Cy5. 환자의 생물학적 시료에 제1항 내지 제9항 중 어느 한 항의 표지된 압타머를 반응시키는 단계,Reacting the labeled aptamer of claim 1 with a biological sample of the patient, 상기 환자의 생물학적 시료에서의 압타머의 결합 정도를 측정하는 단계, 및Measuring the degree of binding of the aptamer in the biological sample of the patient, and 상기 환자의 생물학적 시료에서의 압타머의 결합 정도와 정상 시료에서의 압타머의 결합 정도를 비교하는 단계를 포함하는,Comparing the degree of binding of the aptamer in the biological sample of the patient with the degree of binding of the aptamer in the normal sample, 암 또는 암 전이 진단 정보를 제공하는 방법.A method of providing cancer or cancer metastasis diagnostic information.
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