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研究生： 徐銓龍
Hsu, Chuan-Long
論文名稱： 治療性anti-IgE單株抗體藥物在體外重組模式及體內基因轉殖鼠之藥理作用機制
Pharmacological mechanisms of therapeutic anti-IgE monoclonal body
指導教授： 張晃猷
Chang, Hwan-You
張子文
Chang, Tse Wen
口試委員:
學位類別： 博士
Doctor
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2010
畢業學年度： 98
語文別： 英文
論文頁數： 101
中文關鍵詞： 樂無喘 、抗IgE治療藥物 、免疫複合體 、基因轉殖
外文關鍵詞： Omalizumab, anti-IgE, immune complexes, transgenci mice
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    • 中文摘要
    治療用anti-IgE單株抗體(Omalizumab)是用來中和游離態IgE及減少肥大細胞及嗜鹼性白血球細胞表面高親合性IgE接受器(Fc□RI)的數量，以達到治療第一型過敏性反應的抗體藥物，然而其藥理相關的作用機制仍未被廣範且深入研究。這篇論文利用建立體外重建模式來探討，在病人投與anti-IgE藥物初期所呈現之病程改善的現象與體液中所累積的IgE: anti-IgE免疫複合體的相關性。本研究發現anti-IgE除了可迅速中和游離態IgE外，快速累積的IgE: anti-IgE免疫複合體被證實可結合過敏原。因此，以過敏原特異性IgE組成的IgE: anti-IgE免疫複合體，能在嗜鹼性顆粒白血球細胞膜上的Fc□RI鍵結對過敏原具特異性的IgE形成交叉結合前，先行競爭掉游離態過敏原，進而促成過敏原誘發之過敏反應產生鈍化的現象。
    這篇論文針對另一個本實驗室提出，未被印證的anti-IgE的藥理機制，亦即anti-IgE可否在體內調控膜嵌合型IgE（mIgE）淋巴B細胞(如淋巴纖維母細胞及記憶B細胞)，進而達到阻斷IgE的生合成，作深入研究。我們首先利用單株抗體技術，製備一株結合特異性及藥理特性與omalizumab 相類似之小鼠抗人類 IgE之單株抗體，命名為ASG 16。我們進一步建立C57BL/6 品系之基因轉殖小鼠，其特性為小鼠IgE Fc CH2~CH4區塊被置換成人類IgE CH2~CH4區塊。我們利用此基因轉殖鼠，探討表現mIgE的淋巴B細胞在體內環境下能否被anti-IgE 單株抗體ASG 16所調控。結果顯示，施打ASG 16單株抗體之基因轉殖鼠膜嵌合型m□重鍊RNA轉錄子的合成能被顯著而有效的抑制。體外實驗結果顯示，ASG 16其作用機制是藉由IgG-Fc□R辯識之方式吸引巨噬細胞，以吞噬被ASG 16標定的mIgE B細胞，進而引致抗體導致的細胞抑殺機制（antibody-dependent cellular cytotoxicity , ADCC）達成調控及阻斷IgE的生成。
    膜嵌合型mIgE在RNA選擇性剪接（splicing）的過程中，能產生一種長鏈的膜嵌合亞型IgE，相較於短鏈膜嵌合型IgE，其差異是在鄰近細胞膜表面C端處能表現一段52個氨基酸長的序列(命名為C□mX 區塊) 。比對蛋白質庫及基因庫後，發現此區塊氨基酸排列相當獨特且其僅表現在人類及靈長類之mIgE B淋巴細胞上，相對於短鏈的亞型mIgE，長鏈膜嵌合亞型mIgE表現量又佔優勢。故若以此C□mX區塊做為抗體標靶，並成功發展出循理性設計的單株抗體藥物，則調控mIgE B淋巴細胞即不須先中和體液中的游離態IgE，即可調控膜嵌合亞型IgE淋巴B細胞並進而阻斷其分化成IgE分泌型漿細胞的路徑，最終達到控制IgE合成的效果。這篇論文經過數年建立C□mX基因轉殖小鼠模式，並利用此動物模式做為評估anti-C□mX單株抗體的效用及應用性。

    Abstract
    The therapeutic anti-IgE monoclonal antibodies (mAbs), which have a unique set of binding specificities to human IgE, are designed for neutralizing free IgE and inhibiting IgE production for the treatment of allergic diseases that are caused by type I hypersensitivity reaction. While anti-IgE antibody, omalizumab, has been approved in most major countries to treat patients with severe allergic asthma, the pharmacological mechanisms of anti-IgE have not been well understood. In this thesis research, we have established an in vitro re-constituted model to study the potential contribution of the rapidly accumulated IgE: anti-IgE immune complexes to the efficacious pharmacological effects of omalizumab especially in the initial few weeks after anti-IgE treatment. The results demonstrate that immune complexes comprised of omalizumab and allergen-specific IgE are capable of trapping allergen molecules to inhibit their cross-linking Fc□RI-bound IgE and the consequential activation of the basophils bearing Fc□RI. The results may also explain that some patients respond to omalizumab treatment after the first or second administration of omalizumab and that atopic dermatitis patients with serum IgE many times of 700IU/ml respond well with doses of omalizumab used for patients with IgE below 700IU/ml.
    The other important pharmacological mechanism investigated in this research is whether anti-IgE can modulate mIgE-expressing B cells, including IgE B lymphoblasts and memory B cells, to achieve the ultimate effects of blocking new IgE synthesis. For this goal, we have established a transgenic mouse strain “human IgE Ch2-Ch4 mouse strain”, in which the genomic segment spanning CH2 to CH4 of the mouse own □ immunoglobulin gene has been swapped by the corresponding gene segment of human □ gene by site-specific gene knock-in techniques. We have employed this mouse model to investigate the pharmacological effects of omalizumab-like, mouse anti-human IgE mAb, named ASG 16, on the modulation of IgE-committed mIgE+ B cells in vivo. The results indicate that ASG 16 can inhibit m□ RNA transcription of mIgE B cells in vivo and that ASG 16 can cause antibody-dependent cellular cytotoxicity of mIgE-expressing B cells in the presence of IgG-Fc□R-bearing accessory cells in in vitro culture..
    The human m□ chain, the heavy chain of mIgE, contains a 52-a.a. segment, referred to as C□mX, between CH4 and the C-terminal membrane-anchoring segment. C□mX, which is derived from the alternative splicing of the □ chain RNA transcript, thus provides an attractive antigenic site for the immunological targeting, either by way of a specific monoclonal antibody or a vaccine-like immunogen containing the C□mX domain, of mIgE-B cells for controlling IgE production. Because only humans and Primates species contain C□mX in their mIgE-expressing B cells, this thesis research has endeavored to construct a transgenic mouse strain to insert human C□mX in mouse mIgE. After several years of research, I have constructed “C□mX transgenic mouse strain” for evaluating the efficacy of anti-C□mX mAbs in targeting mIgE-expressing B cells.

    Abstract
    The therapeutic anti-IgE monoclonal antibodies (mAbs), which have a unique set of binding specificities to human IgE, are designed for neutralizing free IgE and inhibiting IgE production for the treatment of allergic diseases that are caused by type I hypersensitivity reaction. While anti-IgE antibody, omalizumab, has been approved in most major countries to treat patients with severe allergic asthma, the pharmacological mechanisms of anti-IgE have not been well understood. In this thesis research, we have established an in vitro re-constituted model to study the potential contribution of the rapidly accumulated IgE: anti-IgE immune complexes to the efficacious pharmacological effects of omalizumab especially in the initial few weeks after anti-IgE treatment. The results demonstrate that immune complexes comprised of omalizumab and allergen-specific IgE are capable of trapping allergen molecules to inhibit their cross-linking Fc□RI-bound IgE and the consequential activation of the basophils bearing Fc□RI. The results may also explain that some patients respond to omalizumab treatment after the first or second administration of omalizumab and that atopic dermatitis patients with serum IgE many times of 700IU/ml respond well with doses of omalizumab used for patients with IgE below 700IU/ml.
    The other important pharmacological mechanism investigated in this research is whether anti-IgE can modulate mIgE-expressing B cells, including IgE B lymphoblasts and memory B cells, to achieve the ultimate effects of blocking new IgE synthesis. For this goal, we have established a transgenic mouse strain “human IgE Ch2-Ch4 mouse strain”, in which the genomic segment spanning CH2 to CH4 of the mouse own □ immunoglobulin gene has been swapped by the corresponding gene segment of human □ gene by site-specific gene knock-in techniques. We have employed this mouse model to investigate the pharmacological effects of omalizumab-like, mouse anti-human IgE mAb, named ASG 16, on the modulation of IgE-committed mIgE+ B cells in vivo. The results indicate that ASG 16 can inhibit m□ RNA transcription of mIgE B cells in vivo and that ASG 16 can cause antibody-dependent cellular cytotoxicity of mIgE-expressing B cells in the presence of IgG-Fc□R-bearing accessory cells in in vitro culture..
    The human m□ chain, the heavy chain of mIgE, contains a 52-a.a. segment, referred to as C□mX, between CH4 and the C-terminal membrane-anchoring segment. C□mX, which is derived from the alternative splicing of the □ chain RNA transcript, thus provides an attractive antigenic site for the immunological targeting, either by way of a specific monoclonal antibody or a vaccine-like immunogen containing the C□mX domain, of mIgE-B cells for controlling IgE production. Because only humans and Primates species contain C□mX in their mIgE-expressing B cells, this thesis research has endeavored to construct a transgenic mouse strain to insert human C□mX in mouse mIgE. After several years of research, I have constructed “C□mX transgenic mouse strain” for evaluating the efficacy of anti-C□mX mAbs in targeting mIgE-expressing B cells.

    目錄 (Table and Content) 目錄 (Table and Content) 1 Chapter I 8 Accumulated immune complexes of IgE and malizumab trap allergens in an in vitro model 8 Summary 9 Background and Rationale 11 Material and methods 15 Cell culture 15 Determining size of IgE:omalizumab IC by size exclusion chromatography 15 Analysis of the binding of IgE:omalizumab IC and antigen by SDS-PAGE 16 Preparation of His-α fusion protein 16 Measuring antigen trapping by IgE:omalizumab IC with ELISA 17 Measuring antigen trapping by IgE:omalizumab IC by fluorescence flow cytometric analysis 18 Measuring activation of RBL.SX38 cells by assaying b-hexosaminidase release 19 Statistical analysis 20 Results 21 IgE and omalizumab form small soluble complexes 21 IgE:omalizumab IC are capable of binding to antigens 21 IgE:omalizumab IC inhibit antigen binding to IgE bound by α subunit of Fc□RI 21 IgE:omalizumab IC inhibit binding of fluorescence-labeled antigen to IgE-saturated basophils 22 IgE:omalizumab IC inhibited antigen-driven sensitization of RBL.SX-38 cells 22 Discussion 24 Figures and tables 28 Chapter II 36 Effects of anti-IgE antibody on mIgE+ B cells in transgenic mice expressing IgE containing human Ig epsilonCH2-CH4 36 Summary 37 Background and rationale 39 Material and methods 42 Cell culture 42 Generation of human Ig□CH2-CH4 transgenic mice 42 Administration of anti-IgE in human Ig□Ch2-Ch4 transgenic mice 44 sIgE and mIgE mRNA expression assayed by Quantitative PCR 45 Preparation and enrichment of CD11b+ macrophages 47 Preparation and enrichment of mIgE+ B cells from human Ig□CH2-CH4 transgenic mice 47 Flow cytometric analysis of Fc□R-dependent ADCC 48 ELISA for free IgE 49 ELISA for total IgE 49 Statistical analysis 50 Results 51 Construction of Ig□ Ch2-Ch4 mIgE+ transgenic mouse 51 Characterization of Ig□ Ch2-Ch4 mIgE+ transgenic mouse 51 Enrichment of human Ig□Ch2-Ch4+/+ mIgE+ B cells in vitro 51 Depletion of mIgE+ B cells by anti-IgE mAb ASG 16 in the presence of CD11b+□ macrophages. 52 Effects of anti-IgE mAb ASG 16 on the down regulation of Ig□ RNA transcript in vivo 53 Discussion 55 Figures and tables 59 Chapter III 71 Development of transgenic mouse strains expressing mIgE containing human CemX domain 71 Summary 72 Background and rationale 74 Materials and methods 77 Animal maintenance and treatment 77 Generation of C□mX-targeting BAC construct 77 Generation of C□mX transgenic mice 79 Genotyping of drug-resistant ES cells and transgenic chimera 80 Results 82 Construction of C□mx transgenic lines 82 C□mX-targeted genotyping 82 Discussion 84 Figures and tables 87 Acknowledgments 95 References 96

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