人類免疫球蛋白E（IgE）在過敏反應中扮演一很重要的角色，導引過敏原激發肥大細胞（mast cells）及嗜鹼細胞（basophils）釋放組織胺等致病化學物質，造成多種過敏症狀。許多藥學研究致力於發展藥物來中和或抑制這兩類細胞所釋放的物質，但這些藥物大多無法有效的治療嚴重性的過敏疾病。1987年張子文博士構想出利用具有結合特異性的單株抗體（anti-IgE）來阻斷IgE引制的過敏機制的方策。此單株抗體已完成三十個以上第二期及第三期的臨床試驗，證實用在治療哮喘、過敏性鼻炎等多種過敏疾病為有效且安全的新藥，並已於美國及歐盟地區核准上市用於治療嚴重性哮喘，臺灣也已在今年四月核准。
此治療性的單株抗體（學名為Omalizumab）最大的特色在於其結合特異性，它能夠結合血液中自由態的IgE，卻不會結合到已經連接在Fc□RI的IgE，以致促成IgE的交聯、Fc□RI的聚集，引發肥大細胞及嗜鹼細胞的激化。先進的分子模擬與蛋白質分子嵌合(molecular docking)技術的結果，估測Omalizumab的結合區位於IgE的CH3區塊，且與Fc□RI的結合位置重疊或相近。雖然IgE與Fc□RI之間的交互作用已經有很多研究，但Omalizumab和IgE確切結合位置以及相接觸的胺基酸仍然未知。
另外，IgE雖是一個由雙硫鍵所連接的對稱性雙分子結構，其與Fc□RI之結合卻呈現不對稱之鍵結。一個Fc□RI分子會同時結合到二個CH3區塊；因此，Fc□RI與IgE成1:1之結合。先前的研究指出，要維持高親和性鍵結的最小IgE單位是一個雙分子性的CH3-CH4區塊。有趣的是，IgE與Omalizumab的鍵結卻又呈現和Fc□RI不同之型態，在接受Omalizumab治療的病人之血液中可以發現IgE與Omalizumab會結合而形成免疫複合體，顯示兩個CH3區塊可分別結合一個Omalizumab分子，且推測一個□ 鏈或一個CH3-CH4應可以單獨具有結合Omalizumab之能力。
本研究利用酵素免疫分析法，證實IgE上單一□鏈的CH3區塊即具有結合Omalizumab之能力。此外亦利用表面電漿共振技術分別測量單一分子的CH3和CH3-CH4區塊及雙分子性的CH3區塊和CH2-CH3-CH4區塊與Omalizumab結合的強度以及其結合動力學特性之比較。研究結果顯示單一分子的CH3區塊與Omalizumab之結合和完整的IgE具有相似的結合親和性。從表面電漿共振的感應圖譜(Sensorgram)也可看出一個雙分子性的IgE分子確實能分別結合兩個Omalizumab分子。綜合以上結果，本研究證實IgE可藉由單一的CH3區塊與Omalizumab進行結合，並且和完整的雙分子性IgE具有同等的結合親和力，並推測和Omalizumab直接接觸之胺基酸應完全位於單一個CH3區塊。

Immunoglobulin E, one of the five classes of antibodies, plays a central role in mediating allergen-induced allergic responses, leading to the sensitization of mast cells and basophils, their discharge of a host of pharmacological mediators, and the manifestation of various allergic symptoms. Active pharmaceutical programs have focused on developing drugs that neutralize or block the mediators released from those two types of inflammatory cells. However, such drugs are often not effective in treating severe allergic diseases. In 1987, Dr. Tse-Wen Chang, who is my thesis supervisor, invented an anti-IgE antibody with a unique set of binding specificities: it binds to free IgE in blood and extravascular space and to membrane-bound IgE on B cells, but not to IgE bound by Fc□RI on mast cells and basophils. One of such antibodies, Omalizumab, has been studied in more than 30 phase II and III clinical trials in various allergic indications and proven to be efficacious and safe in treating allergic asthma, allergic rhinitis, and other allergic diseases. Omalizumab has been approved in the U. S. A., EU, and some other countries for treating moderate-to-severe asthma. It was also approved in Taiwan in past April.
Since the most important property of the anti-IgE therapeutic is its unique binding specificity, it is of great interest to understand the structural aspects in the interaction between anti-IgE and IgE. It is now clear that Omalizumab binds to human IgE on the CH3 domain. Previous molecular simulation and molecular docking studies indicated that the anti-IgE binding site on IgE is adjacent to or overlaps with the Fc□RΙ binding site on CH3. As an IgE molecule is bound by Fc□RI, the binding site for Omalizumab is hindered or masked and can not be bound by Omalizumab. Thus, Omalizumab cannot cross-link IgE bound on Fc□RI and sensitize basophils and mast cells.
While substantial progress has been made in delineating and dissecting the interaction between IgE and Fc□RI, limited knowledge has been obtained regarding the interaction between IgE and anti-IgE. Fc□RI and IgE bind to each other in 1:1 stoichiometry: one Fc□RI molecule can bind to both CH3 domains simultaneously in an asymmetrical manner. In contrast, two Omalizumab molecules can bind to one IgE, and one Omalizumab can bind to two IgE molecules. It has been found that anti-IgE and IgE form 2:1, 1:2, or 3:3 immune complexes in the blood. These results indicate that each □ chain of IgE can distinctively bind to one anti-IgE molecule.
To further analyze the interaction between IgE and Omalizumab, we have expressed various monomeric and dimeric fragments of IgE.Fc and studied their interaction with Omalizumab. The ELISA results indicated that a single CH3 domain on one □ chain can bind to Omalizumab. We also used SPR technique to measure and compare the binding affinities of these IgE.Fc fragments with Omalizumab and their respective binding kinetics. The SPR analysis indicated that Omalizumab binds to a single CH3 domain with affinity comparable to that to intact IgE. The sensorgram also showed that one dimeric IgE molecule can bind to two immobilized Omalizumab molecules while a monomeric CH3 domain can bind to one. These results demonstrated that IgE can bind to Omalizumab through a single CH3 domain with comparable affinity as intact IgE. Furthermore, the results also suggest that the two binding sites on IgE for Omalizumab are independent and that the contact residues of each site should be completely located on one CH3 domain.

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