초록 ▼

• 악성종양이나 급-만성 질환의 치료를 위해 다양한 새 치료법들이 소개되고 있고, 면역 세포를 투여하거나 병변에 특이적으로 결합하여 질병을 치료하는 세포치료술은 난치성 질환의 정복의 가능성을 높여주는 새로운 치료법임. 효율적으로 체내에 투여된 세포의 동태를 평가할 수 있는 분자영상법의 개발에 연구 역량을 집중하였음.

■ 세포치료술의 효능 증대를 위한 방사선 기반 기술개발
[1세부: 세포치료기술의 효능증대를 위한 방사선 복합분자 영상기술 개발]
• 다기능 리포터 유전자를 이용한 항암치료 병행요법과 치료효과 영상

• 악성종양이나 급-만성 질환의 치료를 위해 다양한 새 치료법들이 소개되고 있고, 면역 세포를 투여하거나 병변에 특이적으로 결합하여 질병을 치료하는 세포치료술은 난치성 질환의 정복의 가능성을 높여주는 새로운 치료법임. 효율적으로 체내에 투여된 세포의 동태를 평가할 수 있는 분자영상법의 개발에 연구 역량을 집중하였음.

■ 세포치료술의 효능 증대를 위한 방사선 기반 기술개발
[1세부: 세포치료기술의 효능증대를 위한 방사선 복합분자 영상기술 개발]
• 다기능 리포터 유전자를 이용한 항암치료 병행요법과 치료효과 영상화: MDR1 shRNA와 hNIS 방사성옥소 치료의 병행요법에 의한 다약제 내성 종양 치료 상승효과 평가
• DC 세포를 이용한 면역치료와 hNIS 방사성옥소 치료의 병행요법과 분자영상을 이용한 치료효과 모니터링
• HKII shRNA와 hNIS 방사성옥소 치료를 이용한 미분화 갑상선암의 병행치료 효능 평가 완료
• NIS radioiodine 유전자 치료기법을 이용하여 유방암에 대한 NK 세포의 살상 능력을 성공적으로 증가시켰으며, 뿐만 아니라 두 가지 병행요법에 의한 치료 효능을 영상화함
• 복합분자영상기술을 이용하여 유전자치료/항암제치료/병행치료에 의한 암치료 효능을 성공적으로 평가 및 영상화하였음 (종양 모델: 간암/위암)
• 고감도세포 사멸센서(caspase-3 sensor)를 이용한 세포사멸 영상 및 이를 이용하여 종양치료 효능평가 (NK therapy/Adenoviral gene therapy)
• 생쥐 종양 모델을 확립한 후 방사선 치료, 방사성 핵종 치료와 면역세포치료의 병행 요법, 각종 사이토카인 전처리 등 치료효과를 극대화 할 수 있는 최신 치료 기술 들을 시도하고 이들의 종양치료 효과를 생체 내 영상 시스템을 통해 평가 및 정량화함
[2세부: 방사성 프로브 개발을 통한 세포 추적 및 치료효과 영상화 연구]
• [¹⁸F]FDG와 같은 방사성핵종을 이용해 광학영상을 얻는 새로운 영상기법 개발
• 상피세포 유래 종양에 대한 특이적 선택성을 보이는 펩타이드 1종 개발
• 방광 종양 특이적 펩타이드 (BP) 를 Ga-68로 표지한 후 microPET 영상연구를 수행함
• 신생혈관형성 영상을 위한 ⁶⁸Ga/⁶⁴Cu로 표지된 RGD 유도체를 합성하고 신생 혈관형성에 높은 섭취를 보임을 확인함
• [¹²⁴I]ApoPep 기반의 세포사멸 영상연구를 통한 세포치료효과를 조기에 검증할 수 있음을 확인함
• 소동물에서 ⁶⁴Cu-RGD를 이용한 종양 진단 연구를 성공적으로 진행함
• ApoPep을 Cu-64 표지를 위해 최적의 킬레이트로 MM/DM-TE2A 개발
• ApoPep의 Cu-64 표지를 위해 NOTA 컨쥬게이션된 ApoPep 합성 및 표지
• 종양 특이적 항체의 Cu-64 표지를 위한 킬레이트로 TE2A-Bn-NCS 킬레이트 합성
• 허셉틴 항체를 TE2A-Bn-NCS로 컨쥬게이션 시킨 후 Cu-64로 표지에 성공함
• NOTA 컨쥬게이션 된 ApoPep Cu-64 표지 및 염증모델에서의 생체분포확인
• 항암치료 시 발생하는 세포사멸을 [¹²⁴I]ApoPep-1을 이용한 핵의학 영상을 통하여 확인함
• 종양 특이적 항체의 Cu-64 표지를 위한 킬레이트로 DOTA-Bn-NCS 킬레이트 합성
• 허셉틴 항체를 DOTA-Bn-NCS로 컨쥬게이션 시킨 후 Cu-64로 표지에 성공함
• 종양모델에서 항체를 이용한 생체분포확인 및 핵의학 영상 획득

■ 세포 치료술의 평가를 위한 방사선 기술개발
[1세부: 세포치료기술의 효능증대를 위한 방사선 복합분자 영상기술 개발]
• 방사선 복합분자영상리포터 유전자 시스템 개발: 광학영상 리포터 유전자, firefly luciferase 발현 렌티바이러스 확보, 핵의학영상 리포터 유전자, human sodium iodide symporter gene 발현 렌티 바이러스 확보
• 신개념의 세포 추적 영상술 개발과 함께 새로운 세포 표지제를 개발하고, 방사선 기반 세포 치료술의 효능 증진을 위한 방사선 민감제와 방어제의 개발과 평가를 시행하였음
• 핵의학영상 리포터 유전자를 이용한 대식세포 추적술 개발: NIS 리포터 유전자 발현 macrophage를 확립 하였으며, 대식세포가 염증부위로 이동하는 것을 핵의학영상으로 영상화하였음
• 광학/핵의학 영상리포터 유전자를 이용한 대식세포 추적술 개발: hNIS와 Fluc가 동시에 발현하는 대식세포를 확보하였으며, 광학/핵의학영상을 이용하여 대식세포가 염증부위로 이동하는 것을 성공적으로 영상화하였음
• primary 대식세포에서 핵의학 프로브 ([¹²³I]HIB, [¹⁸F]FDG, ^99mTc-HMPAO)표지 프로토콜 최적화 및 표지효능 비교연구 수행
• 광학영상리포터 유전자를 이용하여 이식된 모유두세포의 생존과 증식을 성공적으로 영상화함
• Cerenkov radiation을 방출하는 I-124, I-131 방사선동위원소를 이용하여 갑상선 및 NIS 발현 세포(정상세포, 암세포)를 광학/핵의학 이중영상을 이용하여 살아있는 개체에서 성공적으로 영상화함
• NIS 리포터 유전자의 새로운 프로프인 [¹⁸F]TFB(tetrafluoroborate)를 이용하여 NIS 발현 암세포 및 종양모델을 성공적으로 영상화함
• 새로운 세포 추적용 복합분자영상용 리포터 유전자 바이러스 시스템 구축
• 수지상세포 (dendritic cell), 자연살상세포 (Natural killer cell), 대식세포(macrophage), 비만세포(mast cell) 등의 면역세포에 리포터 유전자를 전달한 후 리포터 유전자의 활성 측정
• 복합 리포터 유전자가 이입된 면역세포를 체내에 주입 후 이들의 이동 및 생착, 분화 여부를 복합 분자 영상 시스템을 이용해 관찰
• 복합분자영상용 나노파티클을 이용한 면역세포 추적용 프로브 개발 (광학/MRI 이미징 프로브, 핵의학적영상용 이중금나노입자)
• 줄기세포 추적 복합분자영상 및 치료용 벡터확립
[2세부: 방사성 프로브 개발을 통한 세포 추적 및 치료효과 영상화 연구]
• 세포 표지용 방사성 유기 단분자를 매우 효과적으로 표지할 수 있는 방법을 개발하여 [¹²⁴I]HIB를 1mCi 이상 합성
• 세포 표지용 TCL-SPION 나노입자를 I-124로 성공적으로 표지하여 나노입자 사이에 응집 없이 높은 순도로 분리하였음
• 지방유래 줄기세포를 확립하였고 [¹²⁴I]HIB를 이용해 줄기세포도 표지할 수 있음을 확인함
• [¹²⁴I]HIB/OIB와 나노입자를 이용하여 다양한 세포를 표지효율 10% 이상으로 표지
• [¹⁸F]FHBG 1mCi 이상 성공적으로 합성
• 종양에서 추출한 엑소좀을 세포표지자인 [^131/124I]HIB를 이용해 성공적으로 표지하고 분리하였음
• [¹⁸F]TFB의 성공적으로 합성하고 [¹⁸F]TFB가 serum에서 4시간까지 안정함을 확인함
• [^131/124I]HIB를 이용하여 지방유래줄기세포를 심근 주입후 2주간 성공적으로 영상화함
• [^131/124I]HIB를 이용하여 다양한 조성의 리포좀을 표지하였음
• 대식세포가 많이 발현되어 있다고 알려진 CT26모델을 통해 대식세포 섭취여부 규명
• [^131/124I]HIB 표지 리포좀을 이용하여 대식세포 추적 여부를 생체분포확인 및 핵의학 영상을 통하여 확인하였고, 광학영상 및 자기공명에 대한 영상획득
• 핵의학/광학영상을 통하여 염증모델에서도 대식세포의 움직임을 관찰함
[3세부: 암세포 및 면역세포의 반사선 반응 조절]
• 천연물의 radiosensitizer로서의 가능성을 규명하는 연구로서 동물 모델에서암 세포사멸에 대한 효과 검증
• 새로운 미토콘드리아 항산화효소인 IDPm 결여와 IDPm 활성저해 마우스에서 B16F10 melanoma의 암 세포 증식을 검토하여 산화적 스트레스가 관여한 암세포 사멸의 host-tumor micro-environment를 영상기법을 적용하여 규명
• 항암제 내성 유방암의 세포막 NADPH oxidase 발현 및 ROS 발생의 주 장소임을 확인
• 종양미세 환경제어를 통한 효율적 항암치료를 위해 항암제와 혈관신생 억제제의 병용요법이 이루어지고 있으며, 방사선 조사에 의해 혈관신생이 유도된다는 보고에 근거하여 방사선 치료에서 병용할 수 있는 혈관신생 억제제를 검색함
• 혈관신생 억제 화합물의 방사선과 병용요법 시행에서 암세포의 반응 (ROS 발생 및 redox변화)의 구체적 신호전달기전을 규명
(출처:요약서 3~7p)

Abstract ▼

○ Reporter gene system development for nuclear-based multi-modality molecular imaging
• Establishment of reporter gene delivery system to cancer, immune cell using various viral system(hNIS, tk, Fluc, Rflu, and RFP, etc.)
○ Monitoring the therapeutic effects and functional activation of immun

○ Reporter gene system development for nuclear-based multi-modality molecular imaging
• Establishment of reporter gene delivery system to cancer, immune cell using various viral system(hNIS, tk, Fluc, Rflu, and RFP, etc.)
○ Monitoring the therapeutic effects and functional activation of immune cells using multi-modality imaging system in vitro and in vivo
• Reporter gene delivery to cancer, immune cells (macrophage, dendritic cell, etc) using viral system
• Reporter gene expression analysis in infected cells (macrophage and DC)
• In vivo imaging of cancer/immune cell growth and localization using nuclear and optical imaging instruments
• Evaluation of anti-tumor immunity against cancer
• In vivo monitoring of anti-cancer therapeutic effects by single radiation therapy or combined radiation therapy and immune therapy in animal tumor model
○ Non-invasive dual reporter gene imaging for tracking macrophage migration in a murine inflammation model
• Macrophage co-expressing NIS and luc gene was established and the activity of each reporter gene was increased in a cell number dependent manner
• Functional activity such as phagocytosis, cytokine contents and proliferation was not modified after transduction of each reporter gene
• After transfer of reporter macrophage to inflammation mice model, BLI and PET signals was successfully detected in inflamed foci of mice
• Immunohistological analysis with anti-NIS or thy1.1 Ab showed the existence of infused cell in inflamed foci
○ In vivo Monitoring of survival and proliferation of Hair stem cells in a hair follicel generation animal model
• Fluc expressing hair stem cells was established and Fluc assay showed that the Fluc activity was increased in a cell number dependent manner
• Functional activity such as hair formation and proliferation was not modified after transduction of each reporter gene.
• After transplantion of reporter hair stem cell, we successfully monitored the localization and survival of transplanted cells using optical imaging system
• Hair generation was successfully determined following transplantaion of reporter hair stem cells
○ Labeling optimization of primary macrophage using a [¹²³I]HIB, [¹⁸F]FDG, ^99mTc-HMPAO
• When primary macrophage labeled with [¹⁸F]FDG, labeling efficiency is 3% and 20.1% at 4℃ and 37℃, respectively. retention capacity is 64% and 23% at 1h and 5h.
• When primary macrophage labeled with ^99mTc-HMPAO, labeling efficiency is 27.4% and 24.2% at 4℃ and 37℃, respectively. retention capacity is 56.9% and 11.9% at 1h and 5h.
• When primary macrophage labeled with [¹²³I]HIB, labeling efficiency is 18.1% and 17.2% at 4℃ and 37℃, respectively
○ Combined Cerenkov luminescence and nuclear imaging of several radioisotope in vitro and in vivo
• Luminescence signals was increased using a IVIS system when I-131 and I-124 was added to normal FRTL and ARO/NIS cells in vitro
• Bioluminescence signals was detected in thyroid after injection of I-124 to nude mice and animal PET imaging showed intense signal in thyroid
• Bioluminescence imaging and PET imaging showed high uptake in ARO/NIS tumor after I-124 injection
• The decrease of [¹⁸F]FDG uptake was shown in ARO tumor after chemotherapy using bioluminescence and PET imaging
○ Development and evaluation of new tracer for hNIS reporter gene
• When [¹⁸F]TFB is added in Huh/NTG cells, radioactive signals was increased in Huh7/NTG cells but not in parental Huh7 cells
• Bioluminescence imaging showed the high uptake of [¹⁸F]TFB in thyroid of normal nude mice following injection of [¹⁸F]TFB
○ Monitoring of antitumor effects using multimodal imaging system
• The modulation of surface marker was determined in breast cancer cells co-expressing hNIS and Fluc gene after I-131 treatment, resulting increase of killing effect of NK cells against breast cancer cells
• Multimodal imaging showed most effective inhibition of tumor growth in combined group than single treatment group.
• hNIS reporter gene imaging revealed more enhanced inhibition effects of tumor growth in hepatocellular carcinoma mice model treated with combination with GCV plus ANT2 shRNA compared with single treatment.
• Multimodal imaging with optical and radioisotope probe can showed tumor growth inhibition and apoptosis induction in gastric cancer mice model treated with cetuximab, cisplatin and combination
○ Establishment of new multimodal reporter gene imaging system for cell tracking such as immune cells and stem cells
• Successful establishment of two kinds of muti-modal imaging reporter vector
• Validation of respective gene in each reporter vector by luciferase assay, I-125 uptake and FACS analysis
○ In vivo monitoring of the therapeutic effects and activation of immune cell using multi-modality imaging system
• Successful introduction of effluc gene BMDC, NK, macrophage and mast cells
• Determination of reporter activity in infected cells
• primary BMDC: Successful tracking of DC migration to draining lymph nodes with optcial reporter gene
• NK: Successful tracking of NK migration to Cal62 flank tumor model and lung metastasis lesion
• macrophage: in vivo monitoring of reporter macrophage migration to tumor lesion
• mast cell: tracking of mast cell migration to lewis lung cancer lesion
○ In vivo tracking of primary macrophage using multimodal nanoparticle system and intervention study
• In vivo monitoring of macrophage migration as well as evaluation of anti-inflammation drug efficacy was done with FLI/MRI imaging
○ Establishment of double layer gold nanoparticles embedding radionuclide to visualize dynamic cell migration and distribution
• Establishment of radionuclide embedded gold particle
• In vivo tracking of gold particle labelled BDMC in living mice with animal PET/CT
○ In vivo tracking of human amniotic fluid stem cells and evaluation of stem-cell based therapy
• Reporter gene and therapeutic gene was introduced into MSC cells and their migration in vivo was successfully determined with optical imaging system
○ Non-invasive imaging of caspase-3 activation in cancer model by NK based immunotherapy or adenoviral gene therapy (FasL and TRAIL)
• Imaging of caspase-3 activation by NK-based immunotherapy and evaluation of its anti-tumor effect
• Imaging of caspase-3 activation by adenoviral gene therapy and evaluation of its anti-tumor effect
• Imaging of caspase-3 activation by combined treatment (such as combination of NK therapy and chemotherapy or chemotherapy+ NK therapy, etc) and evaluation of its anti-tumor effects
○ Development of new therapeutic modality for cancer treatment and in vivo monitoring of its anti-tumor effects using multimodal imaging system
• Non-invasive imaging of anti-tumor effects by combination of NK therapy and chemotherapy in drug-resistance cancer model
• Non-invasive imaging of anti-tumor effects by NK-based immunotherapy in Cal62/effluc xenograft model and lung metastasis model
○ In vivo monitoring of apoptosis activation using a radiolabeled Apopep
○ Synthesis of [¹²⁴I]HIB (> 1mCi) by development of efficient radiolabeling method of cell-labeling radioactive organic small molecule
○ Successful radiolabeling of TCL-SPION with I-124 and purification method without aggregation of nanoparticles
○ Adipose cell derived stem cell was established and was radiolabeling with [¹²⁴I]HIB
○ Various cells were radiolabeled in higher than 10% yield using [¹²⁴I]HIB/OIB and nanoparticles
○ Development of new optical imaging modality employing radioisotopes such as[¹⁸F]FDG
○ Synthesis of more than 1mCi of [¹⁸F]FHBG
○ Development of epithelial cell origin tumor-specific peptide
○ Imaging study on exosome using [^131/124I]HIB
• [^131/124I]HIB was synthesized in high yield using solvent extraction method
• Exosome was successfully collected from MCF-7 tumor cells
• Exosome collected from tumor cells was successfully radiolabeled with [^131/124I]HIB
• MicroPET imaging study revealed that exosome was transported to mainly liver and spleen
○ Development of tumor imaging agent using [⁶⁸Ga]Y-BP
• Efficient elution method of Ga-68 was established.
• Bladder-specific peptide (BP) was conjugated with NOTA-NCS to give NOTA-BP.
• NOTA-BP was radiolabeled with Ga-68 and successfully purified using HPLC.
• ⁶⁸Ga-NOTA-BP showed high stability in serum and found to be cleared out through kidney based on microPET studies.
○ Synthesis and characterization studies of [¹⁸F]TFB
• [¹⁸F]TFB was successfully radiolabeled with F-18 and purified using simple cartridge.
• -[¹⁸F]TFB showed high stability in serum up to 4h.
○ Synthesis of ⁶⁸Ga/⁶⁴Cu-labeled RGD derivatives for angiogenesis imaging
• RGD was conjugated with NOTA-NCS for ⁶⁸Ga radiolabeling.
• NOTA-RGD showed as high affinity for αvβ₃ as parent RGD itself.
• New TE2A derivatives were developed for efficient radiolabeling of RGD with Cu-64.
• Radiolabeled ⁶⁴Cu-TE2A-NCS-c(RGDyK) showed high tumor uptake in biodistribution studies.
○ Cell trafficking study using [^131/124I]HIB
• Adipose-derived stem cell was radiolabeled in high yield using [^131/124I]HIB
• Radiolabeled stem cell was injected into myocardium and followed up to 2 weeks using PET and SPECT
• The radiolabeled stem cell was successfully differentiated into various cells without any significant cell damage
• [^131/124I]HIB-labeled liposome showed high tumor uptake with very low background signals
○ Apoptosis imaging study using [¹²⁴I/⁶⁸Ga]ApoPep
• ApoPep was conjugated with NOTA and successfully radiolabeled with Ga-68
• Chemotherapeutic effect was evaluated early using non-invasive nuclear imaging studies of [¹²⁴I]ApoPep
○ Angiogenesis imaging study using ⁶⁴Cu-radiolabeled RGD
• New bifunctional chelator was synthesized in order to radiolabel with Cu-64 efficiently
• RGD peptide was conjugated with new BFC and radiolabeled with Cu-64 in high yield
• New blood vessels was successfully imaged in animal tumor models using [⁶⁴Cu]RGD
○ Development of liposome-based imaging probe for macrophage
• Radiolabeling of liposome using [^131/124I]HIB
• Fine-tuning of liposome for efficient macrophage uptake
○ Cu-64 labeled imaging probe for apoptosis detection
• Development of chelator for efficient radiolabeling of ApoPep
• Optimization of radiolabeling condition of ApoPep using Cu-64
○ Imaging probe development based on antibody
• screening of bifunctional chelator for dedicated antibody radiolabeling
• Optimization of antibody radiolabeling condition using Cu-64
○ Nuclear imaging of liposome radiolabeled with [^131/124I]HIB for tracing macrophage and obtaining optical imaging and MRI
○ Optical imaging and nuclear imaging of macrophage in inflammation model
○ Radiolabeling of NOTA-ApoPep with Cu-64 and biodistribution in inflammation model
○ Nuclear imaging of apoptosis induced by treatment of anticancer drug using [¹²⁴I]ApoPep-1
○ Development of chelator (DOTA-Bn-NCS) for efficient radiolabeling of antibody with Cu-64
○ Radiolabeling of antibody conjugated chelator (DOTA-Bn-NCS-Trastuzumab) with Cu-64
○ Biodistribution and nuclear imaging of radiolabeled antibody in tumor model
○ Evaluation of the radiosensitizing effects of natural products, ursolic acid (UA), resveratrol (RV), and xanthohumol on cancer cells
○ Evaluation of radiosensitizing effects of UA and RV in mouse bearing B16F10 melanoma
○ Cell death of cancer cells treated with these natural products exhibited significantly augmented under IR presumably through enhancing the generation of ROS.
○ In adriamycin-resistant human breast cancer cells which also have radioresistance, xanthohumol abolished drug as well as radiation resistance by enhancing expression of multi-drug resistance 1, epidermal growth factor receptor, and signal transducer and activator of transcription 3 in along with suppression of death receptors.
○ The application of natural products as a sensitizer for IR-induced apoptotic cell death offers a promising therapeutic approach for the treatment of cancer.
○ In addition, we also screened chamicals possessing both anti-angiogenic and radiosensitizing ability, because endothelial cells in tumor microenvironment are activated and form new blood vessels by gamma irradiation.
○ Several chemicals including DWMA-003TS and BJ series showed anti-angiogenic and radiosensitizing activity, which, in turn, suppressed cancer cell invasion and adhesion to endothelial cells.
○ Furthermore, cancer cell proliferation was significantly suppressed in B16F10 cells in IDPm KO mouse compared to that in WT mouse under IR, indicating that oxidative stress surrounding cancer cells is critical factor for cancer cell growth and cell death.
○ The similar results were obtained with cells transfected with IDPm anti-sense cDNA and cells treated with oxalomalate, a specific inhibitor of IDPm.
(출처:SUMMARY 22~27p)

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 요약서 ... 3요 약 문 ... 8SUMMARY ... 22CONTENTS ... 28목차 ... 29제1장 연구개발과제의 개요 ... 30 1절. 연구개발의 목적 ... 30 2절. 연구개발의 필요성 ... 312장 국내외 기술개발 현황 ... 37 1절. 국내 기술개발현황 ... 37 2절. 국외 기술개발현황 ... 42 3절. 현기술상태의 취약성 ... 45제3장 연구개발수행 내용 및 결과 ... 47제4장 목표달성도 및 관련분야에의 기여도 ... 276 1절. 목표달성도 ... 276 2절. 관련분야에의 기여도 ... 285제5장 연구개발결과의 활용계획 ... 288 1절. 추가연구의 필요성 ... 288 2절. 타연구에의 응용 ... 289제6장 연구개발과정에서 수집한 해외과학기술정보 ... 292제7장 연구장비의 구축 및 활용 결과 ... 295제8장 참고문헌 ... 296끝페이지 ... 299