【摘要】：嵌合抗體chA21是由本實驗室構(gòu)建的一株抗p185/Her2的工程化人-鼠嵌合抗體,對p185/Her2高表達的腫瘤具有明顯的抑制活性。與另一株已用于臨床的抗p185/Her2抗體藥物Herceptin相比,chA21在抗原親和力和抗體免疫原性方面還有一些不足。為了使其更適用于腫瘤治療,我們基于蛋白結(jié)構(gòu)模擬對chA21進行了親和力改造和人源化改造的研究。 我們通過計算設(shè)計點突變的方法對A21的親和力進行了改造。首先我們通過點突變掃描,精確化了A21的抗原識別表位,并建立了新的抗體互補決定區(qū)(CDR)的分類方法,用這種新方法對抗體A21的各CDR區(qū)內(nèi)的參與抗原識別的位點進行了預(yù)測。在這些分析結(jié)果基礎(chǔ)上,通過計算機分子對接模擬,更精細地揭示了A21和Her2的相互作用模式,并通過計算預(yù)測,設(shè)計了改造抗體A21的抗原親和力的點突變方案并進行了初步實驗驗證。另外,我們結(jié)合分子對接模擬和之前的活性測定結(jié)果,從結(jié)構(gòu)角度闡述了A2l的識別表位與其腫瘤抑制機制的相關(guān)性。 為了降低嵌合抗體chA21的免疫原性,我們通過CDR移植的方法對其鼠源可變區(qū)進行了人源化改造�；贏21可變區(qū)的序列和結(jié)構(gòu)信息,選擇人源化抗體huCC49作為其人源化模板進行抗原結(jié)合區(qū)移植,基于移植之后人化抗體的模擬結(jié)構(gòu),對一些重要的框架區(qū)殘基進行突變方向的篩選,選擇更符合抗體結(jié)構(gòu)穩(wěn)定需要的突變方向,得到A2l的人源化變體H1、H1-1、H1-2的序列。在293T真核表達系統(tǒng)中對Fc融合的人源化變體進行表達,親和力分析結(jié)果表明,與嵌合抗體A21相比,設(shè)計的A21人源化抗體H1、H1-1、H1-2與抗原的親和力相近,并已經(jīng)達到了完全的人源化,具有更好的臨床應(yīng)用前景。
[Abstract]:Chimeric antibody chA21 is an engineering human-mouse chimeric antibody against p185/Her2 constructed in our laboratory. It has obvious inhibitory activity against tumor with high expression of p185/Her2. Compared with another strain of Herceptin, which has been used in clinic, Herceptin has some deficiencies in antigen affinity and immunogenicity. In order to make it more suitable for tumor therapy, we studied the modification of affinity and humanization of chA21 based on protein structure simulation. We modified the affinity of A 21 by calculating the point mutation. First of all, the antigen recognition epitopes of A21 were accurately identified by point mutation scanning, and a new classification method of (CDR) in the complementary determinant region of antibody was established. The sites involved in antigen recognition in each CDR region of antibody A21 were predicted by this new method. On the basis of these results, the interaction model between A21 and Her2 is revealed in more detail by computer molecular docking simulation, and calculated and predicted. A point mutation method was designed to modify the antigen affinity of antibody A 21, and a preliminary experiment was carried out to verify it. In addition, based on the results of molecular docking simulation and previous activity determination, the relationship between the recognition epitopes of A21 and its tumor inhibition mechanism was discussed from the structural point of view. In order to reduce the immunogenicity of chimeric antibody chA21, we humanized the murine variable region by CDR transplantation. Based on the sequence and structure information of A21 variable region, humanized antibody huCC49 was selected as its humanized template for antigen binding region transplantation, based on the simulated structure of humanized antibody after transplantation. The mutation direction of some important frame residues was screened, and the mutation direction which was more suitable for the stability of antibody structure was selected, and the sequence of the human variant H1H1-1H1-2 of A21 was obtained. The human strain of FC fusion was expressed in 293T eukaryotic expression system. The results of affinity analysis showed that compared with chimeric antibody A21, the affinity of the designed human antibody H1H1-1H1-2 to antigen was similar to that of antigen, and it had been completely humanized. It has better prospect of clinical application.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前5條

1 朱娟娟;榮子燁;江冰;劉兢;;抗ErbB2嵌合抗體chA21聚集現(xiàn)象的鑒定和分析[J];生物工程學(xué)報;2008年11期

2 劉志剛,俞煒源;工程抗體的體外成熟[J];生物技術(shù)通訊;2002年01期

3 ;Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185~(c-erbB-2)[J];Cell Research;2003年01期

4 王祥斌,孔健,黃華j;核糖體展示技術(shù)的原理與應(yīng)用[J];中華微生物學(xué)和免疫學(xué)雜志;2002年05期

5 李平,李昀,王琛,劉兢;抗人p185單克隆抗體的體外直接抗癌效應(yīng)[J];中國免疫學(xué)雜志;2002年01期

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