臺灣博碩士論文加值系統

IgE 是一種可引致急性過敏的免疫球蛋白，它是由plasma細胞所分泌，而此種細胞則由含膜IgE（mIgE+）的B細胞分化成。位於人類B細胞表面上的mIgE與分泌型IgE的不同在於C端的部分，其含有細胞膜上，細胞質內片段以及在細胞膜上與CH4區域之間含有一段52氨基酸的片段叫CemX。這段專一於整個已知人類序列的片段可作為指示mIgE+ B細胞之標的。本研究即為開發針對CemX的專一性抗體並評估是否可藉由標的mIgE+ B細胞進而調降IgE的產生。首先我們構建並於動物細胞CHO生產一種含CemX的IgG1.Fc重組蛋白，以此蛋白免疫小鼠並進行細胞融合後得到五株可產生針對CemX單株抗體的融合瘤。ELISA、免疫沈澱法及西方墨點法顯示這些抗體可以鍵結天然的或變性的CemX。競爭性實驗顯示這些抗體會互相抑制彼此對mIgE的鍵結。利用合成的生太篩選發現這五種單株抗體鍵結於同一抗原決定位（epitope），亦即位於CemX C端的RADWPGPP。螢光標示法顯示此種單株抗體可鍵結於一種表現mIgE的B細胞株，SKO-007，並引致細胞表面之斑點狀（patching）以及蓋狀（capping）。我們選定其中一種命名為a20的單株抗體進行功能性分析。生體外的試驗顯示a20可以引致一種表現mIgE.Fc的重組細胞的補體細胞毒殺反應（complement-mediated cytotoxicity, CDC）。生體內的功能性研究則利用一種叫”KM”的基因轉殖小鼠進行，這種小鼠具有包含人類免疫球蛋白基因的染色體片段。這是第一次針對此種小鼠研究其是否可產生抗原專一性IgE及總量IgE，並研究免疫標的mIgE+ B細胞的效應。以ovalbumin (OVA) 免疫此種小鼠發現其血液中含有各種濃度的人類IgE以及針對OVA的專一性IgE。KM小鼠的me鍊含有CemX片段且a20單株抗體可鍵結小鼠內mIgE+ B細胞。生體內試驗顯示當以OVA免疫KM小鼠時a20抗體可抑制小鼠產生針對OVA專一性的人類IgE。由此可知KM小鼠可作為標的mIgE+ B細胞的研究材料。因此我們已製備了針對CemX及標的mIgE+ B細胞的單株抗體，CDC 分析證明此單株抗體具有調降mIgE+ B細胞的潛力，而以KM小鼠進行的生體內分析則證明此單株抗體可抑制對OVA專一性IgE之產生。

IgE is a minute class of immunoglobulin that mediates immediate-type hypersensitivity reactions responsible for various allergic symptoms. IgE is secreted by IgE-producing plasma cells, which differentiate from B cells expressing membrane-bound IgE (mIgE) on the surface. The e chain of human mIgE contains a membrane-anchoring peptide and an extra 52 a.a.-long domain (referred to as CemX) between the membrane anchor and the CH4 domain. CemX is uniquely present in the me chain of human membrane-bound IgE (mIgE) and provides an attractive site for immunological targeting of mIgE-expressing B cells in order to down-regulate IgE level.
This study was designed to evaluate the effects of monoclonal antibodies (mAbs) specific for CemX to target IgE-expressing B cells and decrease IgE production. A CemX-containing IgG1.Fc fusion protein was produced in CHO cells and used to immunize mice; five hybridoma clones secreting mAbs specific for CemX were obtained. Characterization of the mAbs using ELISA, immunoprecipitation, and immunoblotting methods showed that they could bind to both native and denatured forms of CemX. The mAbs exhibited mutual inhibition of binding to mIgE. Epitope mapping using synthetic peptides revealed that all five mAbs recognize the same epitope, RADWPGPP, located near the C-terminus of CemX. Binding of one of the mAbs to mIgE on SKO-007 cells induced the cross-linking of mIgE molecules on the cell surface, resulting in their patching and capping. One of the anti-CemX mAbs, designated a20, was employed for functional analysis. In vitro analysis revealed that the mAb can cause complement-mediated cytotoxicity on mIgE.Fc-expressing transfectants.
In vivo effects were explored with “KM” transgenic mice, which harbor human immunoglobulin miniloci. These mice were first investigated for the profiles of antigen-specific IgE and total IgE production and their suitability for studying the effects of immuno-targeting of IgE-expressing B cells. Human IgE was found to be present over a large range in the blood of KM mice. The mice could produce human ovalbumin-specific IgE when they were immunized with the ovalbumin. The me chain of KM mice contains CemX and mAb a20 could bind to mIgE-expressing B cells. The treatment with a20 inhibited the production of ovalbumin-specific human IgE. The results indicate that KM mice can be employed for studying the immunological targeting of mIgE-expressing B cells. We conclude that anti-CemX mAbs that are specific to human mIgE on B cells have been prepared. The potential of the mAbs for targeting mIgE+ B cells and hence down-regulating IgE was demonstrated by showing that the anti-CemX mAb can inhibit the production of antigen-specific IgE in KM transgenic mice.

Chapter I Background and Significances…………………….…..……………1
Chapter II Monoclonal Antibodies Against The CemX Domain Of Human Membrane-bound IgE And Their Potential Use For Targeting IgE-expressing B Cells……………………………………………...9
Abstract………………………………………………………………10
Introduction…………………………………………..………………12
Materials and methods…………….…………………………………14
Results………………………………………………………………..19
Discussion……………………………………………………………23
Chapter III Inhibition of Antigen-specific human IgE responses in KM transgenic mice by a monoclonal antibody specific for human membrane-bound IgE………………………………………………36
Abstract………………………………………………………………37
Introduction…………………………………………..………………38
Materials and methods…………….…………………………………40
Results……………………………………………………………..…43
Discussion……………………………………………………………45
Chapter IV Discussion and future studies………………………………………54
References…………………………………………………………………...………62

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