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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳彥榮 | zh_TW |
| dc.contributor.advisor | Yen-Rong Chen | en |
| dc.contributor.author | 黃朝暘 | zh_TW |
| dc.contributor.author | Chao-Yang Huang | en |
| dc.date.accessioned | 2023-08-16T16:36:11Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-08-16 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-03 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88974 | - |
| dc.description.abstract | 單株抗體 (mAb) 及其衍生物已成為發展最快速最蓬勃的藥物之一，為了快速篩選出具有治療疾病潛力的人類抗體，本研究規劃建構一個具高度多樣性 (highly diverse) 的合成人類單鏈 (single chain Fv, scFv) 抗體庫。抗體庫中的抗體序列是建構於高穩定性的骨架上，抗體六個 CDR 的設計是模擬天然人類抗體的多樣性，在完成序列優化後進行抗體基因片段合成。此外，為了提高抗體庫的品質，六個 CDR (包括所有不同長度的 CDR-H3) 都先經過 β-內酰胺酶篩選系統篩選，以剔除無法表達抗體的序列，然後重組六個 CDR 成為 scFv 抗體並進行建構抗體庫。在本研究中，完成設計和建構一個具高度多樣性的合成人類抗體庫，命名為 DSyn-1 (DCB Synthetic-1) 抗體庫，此抗體庫之變異區 (variable region) 含有約 2.5E-10 的多樣性。為了評估抗體庫應用於藥物開發的潛力，在研究中規劃針對五種和治療疾病相關的抗原進行抗體篩選的實驗，並進一步以 TIM-3 標的為例，評估 TIM-3 專一性抗體的效力，驗證該抗體庫應用於藥物開發的可能性。噬菌體抗體庫淘選 (panning) 測試的結果顯示，該抗體庫能夠分別針對不同的抗原篩選出數十到上百個專一性抗體。此外，三株 TIM-3 專一性抗體 (DCBT3-4、DCBT3-19 和 DCBT3-22) 在 TIM-3 report assay 中，可顯著抑制 TIM-3 的訊號傳遞，EC50 小於 10 nM，與人類 TIM-3 重組蛋白也有很強的結合力，親和力 (KD) 小於 1 nM。這些結果證實了 DSyn-1 是一個很有潛力的抗體庫，可用於篩選治療用抗體，其篩選出的三株新穎 TIM-3 中和性抗體，也非常有潛力能夠繼續開發為臨床治療用的藥物。 | zh_TW |
| dc.description.abstract | Monoclonal antibodies (mAbs) and their derivatives have become one of the fastest expanding classes of pharmaceuticals. To rapidly isolate potent human antibodies, we have constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library by phage display. The library is designed with high stability scaffolds with six complementarity determining regions (CDRs) tailored to mimic natural antibody compositions. The engineered antibody sequences are optimized for codon usage before synthesis. Furthermore, the six CDRs with variable length CDR-H3s were individually subjected to beta-lactamase selection and then recombined for library construction. In this study, we have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5E-10 transformants. Phage library performance was assessed by panning against five therapeutically relevant antigens including the evaluation of TIM-3-specific antibodies for clinical use. Phage library panning results showed that the library has the potential to generate up to hundreds of specific binders for each target. In addition, three TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity in TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Clone DCBT3-22 was further confirmed to be superior with good physicochemical property and a purity of more than 98% without aggregation. The promising results illustrate the utility of the DSyn-1 library for biomedical research applications and potential of the three novel fully human TIM-3-neutralizing antibodies for further clinical development. | en |
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| dc.description.provenance | Made available in DSpace on 2023-08-16T16:36:11Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 目錄
中文摘要 i 英文摘要 ii 目錄 iii 圖表目錄 v 附錄 vi 第一章 研究背景介紹 1 1-1 單株抗體及其衍生物的發展潛力…………………………………………………………………….1 1-2 人類單株抗體逐漸成為治療用抗體開發的主………………………………….……………..2 1-3 人類單株抗體相關技術平台…………………………………………………..……………………….4 1-4 簡介噬菌體展示技術平台………………….…………………………………………………………….5 1-5 噬菌體展示人類抗體庫的分類……………………………………….……………………………….8 1-6 建構高品質人類抗體庫的要素………………………………………………….…………………….8 1-7 數種和治療疾病相關的抗原………………………………………………………..………………….9 1-8 免疫檢查點……………………………………………………………………………………………….…….11 1-9 TIM-3 單株抗體應用於藥物研發…………………………………………………………………..12 第二章 研究動機與實驗規劃 15 第三章 實驗材料與方法 16 3-1 實驗儀器設備…………………………………………………………………………………………….…..16 3-2 實驗材料…………………………………………………………………………………………………..…….17 3-3 單培養液及試劑配方……………………………………………………………………………..………19 3-4 實驗方法………………………………………………………………………………………………………...20 第四章 實驗結果 28 4-1 DSyn-1合成人類抗體庫CDR序列分析與設計…………………………………….….…..28 4-2 DSyn-1合成人類抗體庫的構建…………………………………………………………….……….29 4-3確認DSyn-1抗體庫的多樣性…………………………………………………….…….……….…..30 4-4進行測試性淘選 (panning)，評估抗體庫應用於篩選對抗原專一性抗體的潛力……………………………………………………………………………………………………..……………….…..31 4-5 驗證抗體庫於製藥的應用…………………………………………………………….……….….…..33 第五章 討論 35 第六章 結論 40 參考文獻 41 圖表 圖一 DSyn-1 人類抗體庫的設計 (1) 48 圖二 DSyn-1 人類抗體庫的設計 (2) 49 圖三 DSyn-1 人類抗體庫的設計 (3) 50 圖四 組裝 scFv 抗體 51 圖五 β-內酰胺酶的篩選系統 52 圖六 DSyn-1 抗體庫的建構流程 53 圖七 評估 DSyn-1 抗體庫的多樣性 (1) 54 圖八 評估 DSyn-1 抗體庫的多樣性 (2) 55 圖九 篩選具有抑制 TIM-3 訊號傳遞能力之抗體 56 圖十 測試 TIM-3 抗體之結合力 57 圖十一 比較 DCBT3-22 和抗體 MBG453 抑制 TIM-3 訊號傳導的能力 58 圖十二 以 SDS-PAGE 和 SEC-HPLC分析純化後的 TIM-3 抗體 59 表一 21個子抗體庫 (經過β-內酰胺酶篩選系統篩選校對) 60 表二 摘要整理五種抗原掏選 (panning) 的結果 61 表三 Phage ELISA against human TIM-3 recombinant protein 62 表四 Phage ELISA against human TGF-β recombinant protein 63 表五 Phage ELISA against human CCR5-peptide 64 附錄 附錄一 IGHV3-23 和 IGKV1-39 的 DNA 以及胺基酸序列 65 附錄二 以 DCB 開發的程式標示出抗體 6 個 CDR 的區域 67 附錄三 將各個製備好的 CDR 片段分別組裝進 scFv template 的示意圖 68 附錄四 建構抗體庫各流程的實驗條件 69 附錄五 長度 3~4 個胺基酸的 CDR-H3 70 附錄六 IgG 抗體表現質體之圖譜 71 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 噬菌體 | zh_TW |
| dc.subject | 合成抗體庫 | zh_TW |
| dc.subject | 單鍊抗體 | zh_TW |
| dc.subject | 治療性抗體 | zh_TW |
| dc.subject | TIM-3 | zh_TW |
| dc.subject | single-chain variable fragment (scFv) | en |
| dc.subject | synthetic antibody library | en |
| dc.subject | antibody therapeutics | en |
| dc.subject | phage | en |
| dc.subject | TIM-3 | en |
| dc.title | 建構巨量合成人類抗體庫應用於抗體藥物開發 | zh_TW |
| dc.title | Construction of a Large Synthetic Human Antibody Library for Therapeutic Antibody Generation | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.oralexamcommittee | 周綠蘋;黃楓婷;侯詠德;趙曉梅 | zh_TW |
| dc.contributor.oralexamcommittee | Lu-Ping Chow;Feng-Ting Huang;Yung-Te Hou;Hsiao-Mei Chao | en |
| dc.subject.keyword | 噬菌體,單鍊抗體,合成抗體庫,治療性抗體,TIM-3, | zh_TW |
| dc.subject.keyword | phage,single-chain variable fragment (scFv),synthetic antibody library,antibody therapeutics,TIM-3, | en |
| dc.relation.page | 71 | - |
| dc.identifier.doi | 10.6342/NTU202302856 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2023-08-07 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| 顯示於系所單位： | 生化科技學系 | |
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|---|---|---|---|
| ntu-111-2.pdf 未授權公開取用 | 2.61 MB | Adobe PDF |
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