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暫不公開

目錄
論文審定書……………………………………………………………………….i
博碩士論文公開授權書.........................................................................................ii
致謝……………………………………………………………………………….iv
中文摘要………………………………………………………………………….v
英文摘要………………………………………………………………………….vi
目錄……………………………………………………………………………….vii
圖次.........................................................................................................................ix
縮寫.........................................................................................................................x
第一章、 研究背景與目的………………………………………………………1
壹、 趨於嚴重的近代社會病症︰骨質疏鬆症…………………………………..1
貳、 治療骨質疏鬆症的方式……………………………………………………..1
參、 利用單株抗體藥物治療骨質疏鬆症………………………………………..1
肆、 全身性的辨認抗原將導致抗體藥物的副作用……………………………..2
伍、 遮蔽抗體的功能區使之變成前驅抗體……………………………………..2
陸、 金屬基質蛋白酶可作為活化抗體的標的…………………………………..4
柒、 開發具蛋白酶活化選擇性之前驅抗體……………………………………..4
第二章、 研究材料及方法………………………………………………………6
(一) 細胞株與細胞培養………………………………………………………….6
(二) 緩衝液與生化試劑………………………………………………………….7
(三) 利用酵素剪切建立含有Hinge的抗體序列……………...………………..8
(四) 利用人類胚胎腎細胞293T初步表現六種Hinge-Prolia抗體……………9
(五) 利用西方點墨法偵測293T細胞表現六種Hinge-Prolia蛋白質的情形…9
(六) 利用Sandwich ELISA偵測293T細胞上清中表現六種Hinge-Prolia蛋白質表現量並利用Prolia®做相對定量…………………………………………10
(七) 利用ELISA評估Hinge屏蔽區抑制Prolia抗體的效果及測試Hinge-Prolia抗體與MMP 9作用後的結合能力………………………………………...11
(八) 利用Expi293細胞大量表現Hinge-Prolia 抗體……...…………………...12
(九) 利用 Protein A Sepharose系統純化Hinge-Prolia 抗體…………………..13
(十) 評估純化後Hinge屏蔽區抑制Prolia 抗體的效果及評估Hinge-Prolia 抗體與MMP 9作用後的結合能力……………………………………………...13
(十一) In Vitro細胞實驗︰以Osteoclastogenesis assay進行功能性測試…...15
(十二) 電腦模擬：以電腦模擬的方式建構出不同的前驅抗體……………..15
(十三) 設計公式以計算前驅抗體的屏蔽效率………………………………..15
(十四) 統計分析………………………………………………………………..16
第三章、 研究成果……………………………………………………………...17
(一) Prolia 和 Hinge-Prolia質體的建構………………………………………..17
(二) 利用人類胚胎腎細胞293T評估Prolia抗體與Hinge-Prolia前驅抗體是否可被表現與分泌……………………………………………………………….17
(三) 評估四種不同的前驅抗體IgG2、IgG4、IgA1及IgA2 Hinge-Prolia前驅抗體屏蔽的效果………………………………………………………………….18
(四) 評估前驅抗體IgG2、IgG4 Hinge-Prolia經由加入蛋白酶 (MMP 9 9s)處理後對於抗原RANKL的結合能力是否回復………………………………….18
(五) 純化前驅抗體IgG2 Hinge-Prolia蛋白質………………………………….19
(六) 評估純化後Hinge-Prolia (IgG2)前驅抗體是否仍具有被MMP 9 9蛋白酶活化的特性………………………………………………………………………..19
(七) In vitro評估Hinge-Prolia與MMP 9 9s作用後是否可以恢復功能並抑制蝕骨細胞 (osteoclast)的生成……………………………………………………..19
(八) 利用電腦模擬預測具有較佳遮蔽效果的前驅抗體………………………..20
第四章、 討論……………………………………………………………………22
壹、 策略︰利用疾病部位過量表現之蛋白酶，開發具有選擇性之前驅抗體..22
貳、 抗體藥物治療可以有效的改善骨質疏鬆症………………………………..22
參、 前驅抗體(Hinge antibody)策略可以適用於任何的抗體…………………..23
肆、 IgG1-G4，IgA1-A2前驅抗體的蛋白表現量亦有差異……………………24
伍、 蛋白酶專一性受質胜肽可隨著疾病做更換以提昇臨床抗體的疾病部位選擇性……………………………………………………………………………..25
第五章、 圖表……………………………………………………………………26
參考文獻…………………………………………………………………………..46

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