臺灣博碩士論文加值系統

為增加抗體疾病區域選擇性以提升治療性抗體藥物的安全性，我們”複製”抗體絞鏈區作為空間性屏蔽之抗體鎖，將其”貼上”於抗體藥物的抗原結合位， 並使用疾病區專一性蛋白酶之受質胜肽連結抗體和抗體鎖，形成前驅抗體, 唯在疾病區才可活化中和疾病區抗原並降低副作用。此篇研究中，我們使用了六種不同的抗體絞鏈區作為抗體鎖屏蔽類風濕性關節炎藥物Remicade (Infliximab)的TNFα結合位，並利用MMP-2/9受質胜肽連結抗體和抗體鎖，形成前驅抗體Pro-Remicade，其中IgG1 抗體鎖屏蔽效果最佳，IgG1 Pro-Remicade之TNFα結合能力較Remicade遠低於395倍之多，而Pro-Remicade經MMP-2/9蛋白酶活化後，其抗原中和能力可完全恢復有效抑制TNFα下游的訊息傳遞路徑。另一方面，IgG1抗體鎖可有效避免抗藥抗體中和前驅抗體pro-Remicade。於小鼠實驗中，Pro-Remicade專一活化於疾病區域(小鼠腳掌)，並與Remicade具有相似的半衰期、生物分布及療效，同時經由伺機性(Listeria infection)感染實驗證實，Pro-Remicade降低了對宿主免疫能力之干擾，相對於Remicade治療之小鼠組別(0%)，Pro-Remicade治療之小鼠組別有更高的存活率(71%)。最後，我們成功了運用空間性屏蔽抗體鎖屏蔽多種臨床抗體藥物。未來此廣泛應用性的抗體鎖或許可為抗體藥物帶來革命性的發展以達到疾病區專一性治療。

To enhance the disease selectivity of antibody for the safety of therapeutic antibody drugs, we “copied” the hinge region as a spatial hindrance-based “antibody lock” and “pasted” it on the antigen binding site of the antibody drug by linking with a disease-specific protease substrate to generate pro-antibody, which can be selectively activated in the disease region and neutralize the antigen in disease region to reduce the side effects. Here, we used six different hinges (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) as antibody lock (Ab lock) to mask the TNFα binding site of rheumatoid arthritis (RA) drug, Remicade (Infliximab) by linking it with matrix metalloproteinase(MMP)-2/9 substrate peptide to generate pro-Remicade. Among the hinge region, IgG1 Ab lock showed the highest potential to remarkably reduce the TNFα-neutralizing ability. The TNFα-binding of IgG1 pro-Remicade was 395-fold weaker than Remicade, and was completely restored after incubation with MMP-2/9 to block TNFα downstream signaling. In addition, the IgG1 Ab lock also significantly prevented the neutralizing effect of an anti-idiotypic antibody. In mice model, pro-Remicade was only selectively activated in the disease site (paw) and it has similar serum half-life, bio-distribution and therapeutic efficacy compared to Remicade, it minimized the interference in the host immunity against Listeria infection, leading a higher survival rate (71%) than that of the Remicade treatment group (0%). Moreover, we demonstrated that the spatial-hindrance-based Ab lock was successfully applied to several antibodies. The universal Ab lock may revolutionize the development of antibody drugs to achieve disease site-specific therapy.

目錄 2
中文摘要 7
英文摘要 8
Abbreviations 9
I. Introduction 10
II. Material and Methods 14
2-1. Cells and Animals 14
2-2. Plasmid construction, expression, and purification of pro-antibody 14
2-3.Analyses the binding affinity of pro-Abs and MMP-2/9 treated pro-Abs by ELISA 15
2-4. Removal of the inhibitory domain from pro-Remicade through MMP-2/9 treatment in vitro 16
2-5. Neutralization of TNFα signal by pro-Remicade 16
2-6. Evaluation of the binding affinity of anti-idiotypic Ab to pro-Remicade 17
2-7. Measurement of the serum half-life and bio-distribution in vivo 18
2-8. Disease-site-specific activation of pro-Remicade in vivo 18
2-9. Therapeutic efficacy of pro-Remicade for rheumatoid arthritis in vivo 19
2-10. Comparison of on-target toxicities of pro-Remicade and Remicade in vivo 20
2-11. Statistical analysis 20
III. Results 21
3-1. IgG1 Ab lock shows the highest masking effect to the antigen binding affinity of Remicade through spatial hindrance 21
3-2. MMP-2/9 treatment can completely remove Ab lock and entirely restore the TNFα binding affinity and neutralizing ability of pro-Remicade 24
3-3 The Ab lock prevents the neutralizing effect of an anti-idiotypic Ab on Remicade 29
3-4 The Ab lock will not change the basic characteristics of Remicade 32
3-5 Site-specific activation of pro-Remicade prevents RA progression 36
3-6 Selective restoration of pro-Remicade reduces risk of opportunistic infection in a human TNFα transgenic mouse model 42
3-7 Spatial-hindrance-based Ab lock can mask the antigen binding affinity of various clinical Ab drugs 46
IV. Discussion 48
V. Conclusion 52
VI. References 53
VII. Appendices 57

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