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研究生:陳國庭
研究生(外文):Kuo-Ting Chen
論文名稱:錸(I)-188三羰基標誌Trastuzumab單株抗體作為乳癌造影及治療劑之研究
論文名稱(外文):Study on [188]Re(I)-Tricarbonyl Labeled Trastuzumab Monoclonal Antibody as Imaging and Therapeutic Agent for Breast Cancer with HER2-overexpression
指導教授:羅建苗李德偉李德偉引用關係
指導教授(外文):Jem-Mau LoTe-Wei Lee
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生醫工程與環境科學系
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:71
中文關鍵詞:錸-188單株抗體
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Herceptin®是近年來常見的抗乳癌藥物,其中的主成分trastuzumab抗體是一種經過基因重組的人類化單株抗體 (humanized monoclonal antibody),其Fab端能辨識HER2,可取代生長因子結合HER2,終止其激酶信號功能,而抑制腫瘤細胞的分化與增生,臨床上用來治療有HER2大量表現的人類乳癌。
錸-188的半衰期長達16.9小時,且能釋放高能的β-粒子 (2.12MeV)及高解析度的γ射線 (0.155MeV),用來標誌單株抗體,可兼具放射免疫治療及放射免疫造影之功效。本研究發展出新的錸-188標誌方式,先以無機化學合成方法在水相中製備錸-188(I)-三羰基離子,[188Re(OH2)3(CO)3]+,再將放射性核種錸-188標誌於trastuzumab單株抗體,作為HER2過量表現乳癌之造影及治療劑。
在本實驗中,以高效能液相層析法、分子大小排阻層析法及蛋白質去性沉澱法測試188Re(I)-trastuzumab的標誌效率及穩定度。並將188Re(I)-trastuzumab注入植有BT-474 (大量表現HER2)或MCF-7 (低表現HER2)腫瘤細胞的實驗裸鼠,經生物分佈及藥物動力學的分析,證實188Re(I)-trastuzumab保有trastuzumab本身的藥物專一性及活性。在SPECT/CT造影的實驗中,188Re(I)-trastuzumab因為與HER2有高度的親和力,使放射性標誌藥物能累積在BT-474腫瘤中,使之顯影。然而,將188Re(I)-trastuzumab注入已植有MCF-7腫瘤的實驗裸鼠,放射性藥物累積於腫瘤的量相對較低,亦無SPECT/CT之顯影。由此研究所得結果可推論188Re(I)-trastuzumab在未來有發展為放射免疫治療藥物之潛力。
Trastuzumab (Herceptin®), a humanized IgG1 monoclonal antibody directed against the extracellular domain of the HER2 protein, acts as an immunotherapeutic agent for HER2-overexpressing human breast cancers. In this study, 188Re, an emitter with 2.12MeV β- and 0.155MeV γ and t1/2 = 16.9h, was labeled to trastuzumab aiming for radioimmunotherapy of HER2/neu-positive breast cancer. The 188Re(I)-tricarbonyl ion, [188Re(OH2)3(CO)3]+, was employed as a precursor for direct labeling 188Re to the monoclonal antibody. The resultant 188Re(I)-trastuzumab was found to be very stable even challenging with histidine. The tumor and normal tissues localization properties of 188Re(I)-trastuzumab in athymic mice bearing BT-474 human breast cancer xenografts (HER2/neu-overexpressing) and bearing MCF-7 human breast cancer xenografts (HER2/neu-low expressing) were investigated. The in vivo biodistribution study demonstrated that 188Re(I)-trastuzumab was specifically accumulated in BT-474 tumor and the image of 188Re localized BT-474 tumor was clearly visualized within the useful lifetime of the radionuclide. By contrast, the 188Re(I)-trastuzumab uptakes in HER2-low expressing MCF-7 tumor was minimal so that the 188Re image localized at the tumor was not seen. It is revealed from the imaging study that 188Re(I)-trastuzumab may be a potential radioimmunotherapeutic agent for treatment of HER2/neu-overexpressing cancers.
第一章、緒論
1.1 放射性分子影像藥物
1.2 放射免疫治療
1.3 ErbB家族及HER2
1.4 Herceptin®
1.5 [188Re(CO)3(OH2)3]+標誌
第二章、實驗材料與方法
2.1 試藥與耗材
2.2 儀器
2.3 [186Re(CO)3(OH2)3]及[188Re(CO)3(OH2)3]+之合成及特性分析
2.3.1 合成步驟
2.3.2 [186Re(CO)3(OH2)3]及[188Re(CO)3(OH2)3]+之分析 2.3.3反應中加入四氫硼酸鹽交換樹脂
2.4 Trastuzumab抗體之純化與定量
2.4.1 實驗步驟
2.5 [188Re(CO)3(OH2)3]+標誌trastuzumab 2.5.1 標誌步驟
2.5.2 188Re(I)-trastuzumab之分析
2.6 [99mTc(CO)3(OH2)3]+標誌trastuzumab
2.7 131I標誌trastuzumab
2.8 活體外實驗
2.8.1 原理
2.8.1.1 細胞受器親和力
2.8.1.2 細胞受器親和力競爭實驗方法
2.8.2 188Re(I)-/ 99mTc(I)-trastuzumab與BT-474細胞受器親和力實驗
2.8.3 188Re(I)-/ 99mTc(I)-trastuzumab與BT-474細胞受器結合競爭實驗
2.9 實驗動物腫瘤模式
2.10 生物分佈
2.11 動物造影
第三章、結果與討論
3.1 [188Re(CO)3(OH2)3]+之製備
3.1.1 反應條件之討論
3.1.2 [188Re(CO)3(OH2)3]+之穩定度
3.1.3 [188Re(CO)3(OH2)3]+與組胺酸配位鍵結合
3.1.4 四氫硼酸陰離子交換樹脂之使用
3.1.5 最佳[188Re(CO)3(OH2)3]+之製備條件
3.2 [188Re(CO)3(OH2)3]+標誌trastuzumab
3.2.1 Trastuzumab抗體之純化與定量
3.2.2 標誌反應條件
3.2.3 188Re(I)-trastuzumab之純化
3.2.4 188Re(I)-trastuzumab之穩定度
3.2.5 188Re(I)-trastuzumab標誌過程中之輻射分解
3.2.6 最佳188Re(I)-trastuzumab之製備條件
3.3 188Re(I)-trastuzumab對HER2之親和力
3.3.1 131I標誌trastuzumab測試藥物活性
3.3.2 188Re(I)-/ 99mTc(I)-trastuzumab之細胞飽和實驗
3.3.3 188Re(I)-/ 99mTc(I)-trastuzumab之細胞競爭實驗
3.3.4 188Re(I)-trastuzumab之特異性鍵結
3.3.5 188Re(I)-trastuzumab對BT-474之親和力
3.4 188Re(I)-trastuzumab之生物分佈及造影
3.4.1 動物模式建立
3.4.2 188Re(I)-trastuzumab之生物分佈
3.4.3 188Re(I)-trastuzumab之SPECT/CT造影
3.4.4 188Re(I)-trastuzumab於動物活體中之藥物動力學 3.4.5 188Re(I)-trastuzumab於生物活體中之特性
第四章、結論
第五章、參考文獻
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