臺灣博碩士論文加值系統

未來藥物發展的世界市場主流，是利用精密的抗體工程技術與重組蛋白量產程序，製造多樣性功能的人體化抗體及抗體Fc融合蛋白，治療人類重要疾病，包括癌症、自體免疫疾病、病毒與細菌感染疾病、過敏性疾病和心臟血管疾病等。這類新藥物如Rituxan (IDEC, San Diego) 是一種治療 B 淋巴癌的嵌合抗體藥物，該藥物藉結合 B 細胞 CD20 分子大量調降 B 細胞數量，自1997 年上市以來，是醫藥市場獲利最高的明星抗體藥物。另一種新上市藥物 Enbrel (Immunex, Seattle)，是將抗體Fc片段與 tumor necrosis factor receptor (TNFR) 融合，透過頡抗作用減少血液中 TNF 濃度，減緩類風濕關節炎症狀，達到治療效果。將人類抗體Fc片段與蛋白質融合製成藥物的優點為：提高蛋白質藥物的構造穩定性，延長藥物在活體的作用時間，在人體的安全性高無排斥現象，易投入工業化製程生產等。
本研究提出全新的治療性疫苗 (therapeutic vaccine) 觀念，將人類 Fc片段與小鼠B細胞表面抗原的胞外胜鏈融合，由人類 Fc 片段突破小鼠免疫系統對自體抗原的相容性，引發抗體產生。這些抗體透過辨識與結合B細胞表面抗原，進而啟動 B 細胞凋亡的控制系統，降低 B 細胞數量，以治療 B 細胞異常活化或增生的疾病，如IgE-引致過敏性疾病與B淋巴瘤等。
我們挑選調控B 細胞生長與分化的表面抗原，包括膜結合分子CD20與B細胞接受器複合體 (B-cell receptor complex) 上的蛋白質單體 Igb。我們將這些蛋白的胞外胜鏈與人類 IgG(g1) 或 IgG(g4) 的Fc片段連結作成融合蛋白免疫小鼠，觀察小鼠體內被誘發的自體免疫反應，是否能調降 B 細胞，進而抑制 B淋巴癌的生長。從實驗結果顯示，人類Fc 融合蛋白能活化小鼠 T 細胞，刺激 B 細胞產生少量自源抗體，使小鼠B細胞數量降低20-40％，削弱小鼠對不相關抗原 chicken ovalbumin 的免疫反應。
綜合上述結論，以B 細胞表面抗原連結外源Fc片斷的疫苗結構設計，能在小鼠成功引發自體免疫反應，調降 B細胞，具有發展成為治療 B 細胞異常活化或增生疾病的藥物潛力。

One major area in pharmaceutical development in the coming decade will center on antibody-engineering technology and large-scale manufacturing of recombinant proteins. The leading products include humanized antibodies and immunoglobulin Fc fusion proteins designed to specifically target crucial etiologic or virulent factors for treating patients with a variety of diseases, e.g. cancer, autoimmune diseases, bacterial and viral infection, allergy, cardiovascular diseases, etc. For example, Rituxan, a chimeric antibody for treating B-lymphoma, binds to CD20 molecules on B cells leading to apoptosis and cell lysis mediated by antibody-dependent cell cytotoxicity and complement-dependent cytotoxicity. Another drug is Enbrel, used for treating rhumatoid arthritis, is a fusion protein of Fc and the extracellular portion of TNF receptor, capable of neutralizing TNF.
The rationale for linking Fc fragment of IgG with therapeutic proteins or peptides to form fusion proteins are based on the following potential advantages: Fc fragments provide the dimeric conformation that sometimes enhances the stability of linked partners; improving the pharmacokinetic properties of the therapeutic partners in vivo; Fc may bind to Fc receptors and elicit immune mechanisms; amenable to large-scale manufacturing.
In this thesis, we are experimenting in mouse models a novel rational-drug-design concept in designing a new version of therapeutic vaccine. Several surface proteins unique to mouse B cells, to which immune response was to be specifically induced, were fused with human IgG1.Fc fragment. In addition to the potential benefits mentioned above, the Fc fragment was used to provide T helper cell-reactive peptides in order to lift the immune tolerance towards self-antigen.
We have selected two B cell-specific proteins, CD20, a component of a calcium-channel, and Igb, a part of the B cell antigen receptor complex, for the present studies. CD20 is responsible for regulating B-cell proliferation and Igb is involved in signal transduction coming through the receptor. We fused the extracellular segments of mouse CD20 and Igb to human Fc fragment of immunoglobulin g1 or g4 and used them to immunize BALB/c mice. In those immunized mice, auto-reactive antibodies were induced and the proportions of B cells among lymphocytes in the peripheral blood declined 20-40%. The weakened B-cell activity was reflected in the observation that in those immunized mice the immune response towards a bystander irrelevant antigen chicken ovalbumin was significantly reduced.
In conclusion, the results of the present studies have shown in mouse models that specific autoreactivity against self-antigens on B cells can be elicited by immunizing with components of self proteins linked to Fc fragment of IgG. The results warrant further studies to the new approach of developing therapeutic vaccines for the treatment of B-cell-related diseases.

Chinese abstract
English abstract
Acknowledgements
Catalogue
Chapter 1. Rationale of the research : background and significance
Chapter 2. Down regulation of B cells by immunization with a fusion protein of a self CD20 peptide and a foreign IgG.Fc fragment
Experimental approach
Results
Discussion
Chapter 3. Inducing specific reactivity against B cells in mice by immunizing with an Fc fusion protein containing self-Igb
Experimental approach
Results
Discussion
Chapter 4. Monoclonal antibodies against CemX domain in human membrane-bound IgE and their activity on depleting IgE-expressing B cells
Experimental approach
Results
Discussion
Conclusion
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