發(fā)布時間：2018-05-03 08:43

  本文選題：CD20 + 嵌合抗體��； 參考：《河北大學(xué)》2007年博士論文

【摘要】： CD20是人B淋巴細(xì)胞表面特有的標(biāo)志,它表達(dá)在正常人B淋巴細(xì)胞表面,但在多數(shù)惡性B淋巴細(xì)胞表面也呈高表達(dá),因此它是治療B細(xì)胞型非霍奇金淋巴瘤(Non-Hodgkin's lymphoma, NHL)的理想靶抗原�；诒卷椖拷M前期研究獲得CD20嵌合抗體TGLA (IgG1κ)的基礎(chǔ)上,本論文通過特異性靶向結(jié)合、親和力分析、B淋巴瘤細(xì)胞的體外殺傷效應(yīng)和荷瘤動物的體內(nèi)抑瘤等實驗,評價了TGLA嵌合抗體的生物學(xué)活性；此外,本論文還利用計算機(jī)生物信息學(xué)的抗原抗體立體結(jié)構(gòu)模建分析系統(tǒng),對一株具有生物學(xué)活性的抗CD20鼠源抗體1-28(IgM),進(jìn)行了人源化改造,分別構(gòu)建了兩種融合蛋白形式的嵌合抗體(IgG型),并分析比較了人q1區(qū)對于改造后的工程抗體生物活性的影響。 1.抗CD20嵌合抗體TGLA的體內(nèi)、外生物活性研究 免疫共沉淀的結(jié)果證實：抗CD20嵌合抗體TGLA可特異性的沉淀出CD20蛋白；并且間接熒光分析的結(jié)果進(jìn)一步表明TGLA可與CD20+細(xì)胞系特異性結(jié)合。補(bǔ)體依賴的細(xì)胞毒性作用(complement-dependent cytotoxicity, CDC)和抗體依賴性細(xì)胞介導(dǎo)的細(xì)胞毒性作用(antibody-dependent cell-mediated cytotoxicity, ADCC)分析證明：TGLA能夠特異性地殺傷惡性B淋巴細(xì)胞；應(yīng)用TGLA治療荷瘤裸鼠,平均存活期延長了1.7倍。 2.抗CD20抗體1-28的結(jié)構(gòu)模擬、改造及結(jié)合活性檢測 在計算機(jī)模擬基礎(chǔ)上,把鼠源抗CD20抗體1-28(IgM)的輕、重鏈可變區(qū)基因和人IgG1的Fc或完全的重鏈恒定區(qū)融合,分別構(gòu)建了兩種工程抗體LH23(VL-Linker-VH-Hγ-Cγ2-Cγ3)和LH1-3 (VL-linker-VH-Cγ1-Hγ-Cγ2-Cγ3)。把構(gòu)建的表達(dá)載體瞬時轉(zhuǎn)染293T細(xì)胞后,通過western blot證實兩種工程抗體均可被抗人IgG抗體所識別；FACS分析證實兩種工程抗體都可與CD20+細(xì)胞系特異性結(jié)合,其中LH1-3與CD20+細(xì)胞系Daudi、Raji細(xì)胞結(jié)合活性明顯高于LH23,與同源模建、分子力學(xué)方法預(yù)測的結(jié)果一致。這些實驗結(jié)果充分說明人q1區(qū)的剛性結(jié)構(gòu)對于由IgM型改造成的IgG型抗體是十分關(guān)鍵的。
[Abstract]:CD20 is a unique marker on the surface of human B lymphocytes. It is expressed on the surface of normal human B lymphocytes, but it is also highly expressed on the surface of most malignant B lymphocytes. Therefore, it is an ideal target antigen for the treatment of non-Hodgkin 's lymphoma (NHLL) of B-cell non-Hodgkin 's lymphoma. Based on the previous study of CD20 chimeric antibody TGLA G 1 魏, this paper analyzed the killing effect of B lymphoma cells in vitro and tumor inhibition of tumor-bearing animals by specific targeting binding and affinity analysis. The biological activity of TGLA chimeric antibody was evaluated, in addition, a bioinformatics antigen-antibody stereoscopic structure model analysis system was used to humanize a strain of anti-IgM antibody 1-28 CD20, which had biological activity. Two types of chimeric antibodies were constructed, and the effects of human Q1 region on the bioactivity of engineered antibodies were analyzed and compared. 1. In vivo and in vitro bioactivity of anti CD20 chimeric antibody TGLA The results of co-immunoprecipitation showed that TGLA, an anti CD20 chimeric antibody, could specifically precipitate CD20 protein, and indirect fluorescence analysis showed that TGLA could specifically bind to CD20 cell line. The cytotoxic effects of complement dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCCC) showed that TGLA could specifically kill malignant B lymphocytes, and the average survival time of TGLA was 1.7 times longer than that of normal mice. 2. Structural Simulation, Modification and binding activity Detection of Antibody 1-28 against CD20 On the basis of computer simulation, two engineering antibodies, LH23(VL-Linker-VH-H 緯 -C 緯 2-C 緯 3) and LH1-3 VL-linker-VH-C 緯 -H 緯 -C 緯 -C 緯 3, were constructed by fusion of the light and heavy chain variable region genes of mouse anti-IgM antibody 1-28 CD20 with FC or complete heavy-chain constant region of human IgG1. The two engineering antibodies, LH23(VL-Linker-VH-H 緯 -C 緯 2-C 緯 3 and LH1-3 mAb VL-linker-VH-C 緯 1-H 緯 -C 緯 -C 緯 3, were constructed respectively. After transient transfection of the constructed expression vector into 293T cells, western blot confirmed that the two kinds of engineering antibodies could be recognized by anti-human IgG antibodies and confirmed that both engineering antibodies could specifically bind to CD20 cell lines. The binding activity of LH1-3 to CD20 cell line Daudio Raji was significantly higher than that of LH23, which was consistent with the results predicted by molecular mechanics method. These results fully show that the rigid structure of human Q1 region is very important for the transformation of IgG type antibody from IgM type.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 聶靜苑;劉煜;;人源單克隆抗體藥物質(zhì)量控制與分析[J];中國生化藥物雜志;2012年02期

，

本文編號：1837820