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研究生(外文):Shiung, Yu-Yu
論文名稱:Non-anaphylactic anti-IgE monoclonal antibodies capable of binding to IgE on its low-affinity receptors
指導教授(外文):Chang, Tse WenPan, Rong-Long
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IgE is a central mediator responsible for immediate-type hypersensitivity reactions. The anti-IgE monoclonal antibody (mAb), omalizumab, has been shown in numerous clinical trials to be efficacious in the treatment of severe allergic asthma and other allergic diseases. Omalizumab was designed to bind to free IgE in blood and membrane-bound IgE (mIgE) on B cells, but not to IgE bound by the high-affinity IgE Fc receptors (Fc?悐I) on basophils and mast cells and by the low-affinity IgE Fc receptors (CD23) on B cells and many other cell types. Additionally, by binding to free IgE, omalizumab can prevent it from binding to both Fc?悐I and CD23. Omalizumab binds with high affinity to a site on CH3 domain of ? chain, which overlaps or is adjacent to those for Fc?悐I and CD23, and therefore cannot bind to IgE already bound by Fc?悐I and CD23. While it is understood that a therapeutic anti-IgE mAb must not bind to IgE on Fc?悐I, the requirement and benefits for it to not to bind to IgE on CD23 is not convincing.

Studies by other groups have shown that CD23 molecules on the cell membrane are unstable and are cleaved into a secreted form, sCD23, and that the binding by IgE or anti-CD23 mAbs stabilizes CD23 on the cell membrane and induces inhibitory signals leading to the down-regulation of IgE synthesis. It was also found that interfering CD23 function, such as by cross-linking CD23 on B cell surface, can cause the down-regulation of IgE production. In this thesis, we have developed anti-IgE mAbs that mimic omalizumab in various aspects except their ability to bind to IgE on CD23. These newly developed mAbs bind to conformational epitopes on IgE. They do not bind to IgE on Fc?悐I and thus do not induce the degranulation of IgE-pulsed rat basophilic leukemic cells (RBL SX-38), which had been transfected with the genes of human Fc?悐I. One of these anti-IgE mAbs, 8D6, does not induce the degranulation of these cells at various concentrations. However, these mAbs can bind to IgE bound by CD23 on B cells and may cross-link IgE-bound CD23 molecules. The ability of the mAbs to cross-link IgE-occupied CD23 on the cell surface of B cell lines is being tested. The results suggest that these anti-IgE mAbs may render a set of pharmacological mechanisms, which are somewhat different from that of omalizumab, for controlling IgE in patients with allergic diseases.

對於急性過敏反應來說,IgE是一個重要的媒介物。在許多的臨床試驗中,omalizumab (樂無喘)這種抗IgE的單株抗體,已被證實能夠有效地治療嚴重的過敏性哮喘以及其它過敏性疾病。Omalizumab被設計為能與血液中之未被佔用的IgE (free IgE)以及與B細胞表面之膜鑲嵌型IgE (mIgE)做結合;而不能與嗜鹼性球細胞(basophils)或巨大細胞(mast cells)表面之高親和力IgE受器(Fc?悐I)所抓住的IgE結合,同時也不能與B細胞或許多其它類細胞的表面之低親和力IgE受器(CD23)所抓住的IgE結合。除此之外,omalizumab可藉由結合未被佔用的IgE,而阻止它們與Fc?悐I或CD23結合。Omalizumab能夠以高親和力結合於 ? chain之CH3區域的一個部位,且此部位重疊或鄰近於Fc?悐I及CD23所結合的部位,進而才使得omalizumab無法結合至已被Fc?悐I或CD23抓住的IgE。對於一個具有療效的抗IgE單株抗體來說,它絕不能結合至Fc?悐I抓住的IgE;然而,目前尚無證據能證實,避免結合至CD23抓住的IgE是否為必須且有益處的。

根據其它團隊的研究結果顯示,在細胞膜表面的CD23分子是不穩定的,而且會被剪切為一種可溶的CD23 (soluble CD23, sCD23);此外,IgE或抗CD23的單株抗體結合至細胞膜上的CD23分子之後,能夠使它們變得穩定,並且引致抑制性的訊號,進而調降IgE之合成。也有人發現若去干預CD23的功能,例如將B細胞表面的CD23做交叉結合(cross-linking)就能夠調降IgE的產生。在這篇論文中,我們發展了一些抗IgE的單株抗體,這些抗體的許多性質都與omalizumab相仿,不同之處在於它們能夠與CD23抓住的IgE結合。這些新發展出來的單株抗體會結合至IgE分子上的構形式抗原決定區,而且由於它們不會結合至Fc?悐I上的IgE,因此對於以人類Fc?悐I基因所轉殖的大鼠嗜鹼性球細胞瘤(rat basophilic leukemic cells, RBL SX-38 cells)來說,即使細胞表面的Fc?悐I被IgE佔滿,這些抗體仍不會造成「去顆粒作用」 (degranulation)。而在這些抗體之中,8D6即使在不同濃度之下,仍不會造成這些細胞的去顆粒作用。然而,這些抗體能結合至B細胞表面之被CD23抓住的IgE,因此可能使得這些抓住IgE的CD23進行交叉結合。我們目前在測試的是,對於B細胞表面之被IgE佔據的CD23來說,這些抗體是否有能力使它們交叉結合。現有的實驗結果顯示,這些抗體在控制過敏病人體內之IgE的方面,可能具備某些不同於omalizumab的藥理機制。

誌謝 1
中文摘要 2
Abstract 4
Abbreviations 5
Introduction :
Rationale and significances 6
Materials and methods
1. Cell cultures 8
2. Preparation of mIgE.Fc recombinant proteins 10
3. Preparation of peroxidase-conjugated omalizumab 11
4. Generation of anti-IgE mAbs 12
5. Preparation of omalizumab-coupled CNBr-activated resin 14
6. Purification of human IgE and anti-IgE mAb 15
7. Production of recombinant proteins 16
8. ELISA tests 18
9. Preparation of IgE:anti-IgE immune complexes 20
10. Flow cytometry analysis 21
11. Sensitization of RBL-SX38 cells and ??hexosaminidase release assay 22
1. Generation of anti-human IgE mAbs that bind to IgE on Fc?悐II but not Fc?悐I 23
2. The mAb 8D6 competes with omalizumab for binding to IgE 23
3. Inability of 8D6 to bind to IgE on basophils 24
4. Inability of 8D6 to bind to IgE-saturated recombinant ??nchain of Fc?悐I 25
5. The mAb 8D6 blocks the binding of IgE to Fc?悐I 25
6. The mAb 8D6 can bind to IgE-pulsed SKW cells 25
7. 8D6 can bind to IgE on recombinant CD23 26
8. The immune complexes of 8D6 and IgE bind to B cells expressing CD23 26
9. Inability of 8D6 to sensitize RBL SX-38 cells 27
Discussion 28
Figures 31
Appendix 38
References 51

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