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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳漢忠(Han-Chung Wu) | |
dc.contributor.author | Mei-Ying Liao | en |
dc.contributor.author | 廖美英 | zh_TW |
dc.date.accessioned | 2021-06-08T00:53:48Z | - |
dc.date.copyright | 2015-09-24 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-05-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18179 | - |
dc.description.abstract | 在本篇研究中,我們使用口腔癌細胞作為抗原,來免疫BALB/c老鼠,利用融合瘤技術製備出十二株之單株抗體,其中一株OCAb9-1抗體,能專一性對抗頭頸部癌細胞,對正常細胞則没有反應。根據免疫親和性層析 (immunoaffinity chromatography),質譜儀分析,免疫共沈澱法(co-immunoprecipitation)和核糖核酸干擾 (RNA interference)的研究,證明OCAb9-1抗體的標的蛋白是上皮細胞黏著分子(EpCAM)。上皮細胞黏著分子(EpCAM)大量表現在上皮細胞癌,與促進癌細胞惡化有顯著關係。過去文獻研究EpCAM高度表現在一些具癌症起始能力之癌細胞或腫瘤幹細胞。我們實驗室研究團隊也證明EpCAM會影響癌細胞的轉移能力、自我更新和起始能力。近年來,多篇研究運用EpCAM做為標的蛋白,發展偵測血液中循環的癌細胞或腫瘤幹細胞。為了發展治療性抗體,利用抗體純化大腸直腸癌細胞上的EpCAM蛋白為抗原再次免疫老鼠,並且建立新的五株對抗EpCAM的單株抗體,其中發現EpAb2-6能引起癌細胞凋亡,並且抑制腫瘤生長。治療性抗體EpAb2-6與大腸直腸癌臨床化療藥物irinotecan, fluorouracil, and leucovorin (IFL)或胰臟癌臨床化療藥物gemcitabine結合後,能有效增進治療效果及延緩轉移,減少治療期間動物體重下降情形,並且有效延長動物存活率。為了避免人體對鼠源抗體的免疫排斥反應,進一步利用擬人化抗體技術將EpAb2-6改造成適於人類臨床使用的人源性抗體。改造後的抗體除了保有誘發腫瘤細胞凋亡的能力外,亦不影響其對蛋白的親和力,研究成果提供未來癌症治療的新策略。本研究製備之anti-EpCAM單株抗體,除了可以直接造成腫瘤細胞的凋亡外,具有良好的專一性及親和力,未來則相當有潛力應用於臨床癌症診斷、預後、標靶治療和醫學影像。 | zh_TW |
dc.description.abstract | We have newly generated 12 monoclonal antibodies (mAbs) including OCAb9-1, which specifically binds to HNSCC but not to normal cells. According to the results from immunoaffinity chromatography, LC-MS/MS analysis, co-immunoprecipitation, and RNA interference studies, the target protein of OCAb9-1 is epithelial cell adhesion molecule (EpCAM). Epithelial cell adhesion molecule (EpCAM) is known to be overexpressed in epithelial cancers associated with enhanced malignant potential, particularly colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC). However, it is yet to be determined whether targeting EpCAM can directly inhibit the progression of malignance. For development of therapeutic antibodies, we newly generated five EpCAM mAbs. One of these EpCAM mAbs, EpAb2-6, was found to induce cancer cell apoptosis in vitro, inhibit tumor growth, and prolong the overall survival of mice with human colon carcinoma xenografts and pancreatic cancer metastatic animal model. EpAb2-6 also increases the therapeutic efficacy of irinotecan, fluorouracil, and leucovorin (IFL) therapy in colon cancer animal model and gemcitabine therapy in pancreatic cancer animal model. Investigation of the mechanism by which EpAb2-6 inhibits tumor growth revealed that EpAb2-6 binds to cancer cells, whereby it exerted cytotoxic activity by inducing apoptosis pathway and blocks EpCAM intracellular domain (EpICD) cleavage, thus preventing the nuclear localization of EpICD. Furthermore, EpAb2-6, which binds to positions Y95 and D96 in the EGF-II/TY domain of EpCAM, inhibits production of EpICD, thereby decreasing its translocation and the subsequent signal activation. EpAb2-6 was successfully converted to a humanized version (hEpAb2-6) that retained cancer killing activity induction of cancer cell apoptosis. Collectively, our results indicate that the novel EpCAM mAb can potentially be used for cancer-targeted therapy. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:53:48Z (GMT). No. of bitstreams: 1 ntu-104-D95422002-1.pdf: 6495683 bytes, checksum: b4ac98a7308be58e3c72531b234994ce (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | Contents 口試委員會審定書………………………………………………………………. …..2 誌謝……………………………………………………………………...…………….3 中文摘要……………………………………………………………………………....4 Abstract……………………………………………………………………………….5 Contents……………………………………………………………………………….7 Content of table ………………………………………………………………….....10 Content of figures …………………………………………………………………..11 List of abbreviations ………………………………………………………………..12 Chapter I: Introduction…………………………………………………………….14 1. Head and neck squamous cell carcinoma and colon cancer………………...…14 2 Antibody therapy of cancer…………………………………………………….15 3. Epithelial cell adhesion molecule (EpCAM)…………………………………..16 3.1 Structure of EpCAM……………………………………………………..17 3.2 Oncogenic signaling of EpCAM ………………………………………...18 3.3 EpCAM-targeted therapies……………………………………………….20 3.3.1 Clinical experience with edrecolomab …………………………….21 3.3.2 Clinical experience with adecatumumab …………………………..22 3.3.3 Development of EpCAM-targeted mAbs ………………………….24 Chapter II: Materials and Methods………………………………………………..27 2-1 Cell lines and culture ………………………………………………………..27 2-2 Generation of monoclonal antibodies recognizing cancer cells and purification of IgG ……………………………………………………………….……….28 2-3 ELISA…………………………………………. ……………………………28 2-4 Western blot analysis ……………………………………………………...…29 2-5 Immunofluorescent staining …………………………………………………29 2-6 Flow cytometry ……………………………………………………………...30 2-7 Identification of the target protein ……………………………………..……30 2-8 Immunoprecipitation and Western blot analysis ………………….…………31 2-9 Immunohistochemistry………………………………………………………32 2-10 Lentivirus production ………………………………………………………33 2-11 RNA extraction and quantitative real-time RT-PCR ………………….……33 2-12 Surface plasmon resonance ………………………………………...………34 2-13 Generation of monoclonal antibodies recognizing EpCAM ………….……34 2-14 Apoptosis assays ……………………………………………….…………..35 2-15 MTT assay …………………………………………………..……………..35 2-16 Non-attachment cell death assay ………………………………..……….…36 2-17 Identification of B-cell epitopes of EpAb2-6 by phage display ……….…..36 2-18 Use of EpCAM mutants to identify the B-cell epitope of EpAb2-6 ……….37 2-19 Construction of EpCAM deletion mutant ………………………….………37 2-10 Synthesis of EpAb2-6-HiLyte-750 conjugated (EpAb2-6-HL750) and imaging ………………………………………………………………….....37 2-21 Animal model for analysis of antitumor efficacy ………………….….……38 2-22 Colon and pancreatic cancer metastatic animal model ……………...……..39 2-23 Cloning and CDR sequencing of anti-EpCAM antibodies ………………...40 2-24 Construction and expression of humanized EpAb2-6 …………………….40 2-25 Statistical analysis ………………………………………………………….41 Chapter III: Results ………………………………………………………..………42 3-1 Generation and characterization of mAbs recognizing cancer cells …...……42 3-2 Inhibition of cancer cell growth in vitro by EpCAM shRNA ……………….43 3-3 Generation and characterization of mAbs recognizing EpCAM …………....43 3-4 The EpAb2-6 antibody inhibits the growth of cancer cells …………...…….44 3-5 Identification of B cell epitopes of EpAb2-6 ……………………….………45 3-6 In vivo tumor targeting of anti-EpCAM mAbs ………………….………….46 3-7 Combinatorial treatment of human colon carcinoma xenografts with EpAb2-6 and IFL …………………………………………………...………………….47 3-8 EpAb2-6 increases the survival rate of mice in colon and pancreatic cancer metastatic animal models ……………………………………………...…….49 3-9 Development of humanized EpAb2-6 (hEpAb2-6) and hEpAb2-6 antibody inhibits the growth of cancer cells ……………………………………..……51 Chapter IV: Discussion and conclusion ……………………………...……………53 Acknowledgement …………………………………………………...……………..61 References …………………………………………………………………………..62 Tables ……………………………………………………………….………………74 Figures……………………………………………………………………………….79 | |
dc.language.iso | en | |
dc.title | 研發新穎對抗EpCAM的單株抗體運用於癌症之治療與診斷 | zh_TW |
dc.title | Novel anti-EpCAM monoclonal antibodies for the development of diagnostic and therapeutic applications | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 郭彥彬(Yen-Ping Kuo) | |
dc.contributor.oralexamcommittee | 廖永豐(Yung-Feng Liao),高承福(Cheng-Fu Kao),呂仁(Joyce Jean Lu) | |
dc.subject.keyword | 大腸癌,EpCAM,治療性抗體,醫學影像,癌症治療, | zh_TW |
dc.subject.keyword | Colorectal carcinoma,EpCAM,therapeutic antibody,targeting imaging,cancer therapy, | en |
dc.relation.page | 102 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2015-05-14 | |
dc.contributor.author-college | 牙醫專業學院 | zh_TW |
dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
顯示於系所單位: | 臨床牙醫學研究所 |
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