抗CD20抗体的改造及其生物活性研究

发布时间：2018-05-03 08:43

本文选题：CD20 + 嵌合抗体　；参考：《河北大学》2007年博士论文

【摘要】： CD20是人B淋巴细胞表面特有的标志,它表达在正常人B淋巴细胞表面,但在多数恶性B淋巴细胞表面也呈高表达,因此它是治疗B细胞型非霍奇金淋巴瘤(Non-Hodgkin's lymphoma, NHL)的理想靶抗原。基于本项目组前期研究获得CD20嵌合抗体TGLA (IgG1κ)的基础上,本论文通过特异性靶向结合、亲和力分析、B淋巴瘤细胞的体外杀伤效应和荷瘤动物的体内抑瘤等实验,评价了TGLA嵌合抗体的生物学活性；此外,本论文还利用计算机生物信息学的抗原抗体立体结构模建分析系统,对一株具有生物学活性的抗CD20鼠源抗体1-28(IgM),进行了人源化改造,分别构建了两种融合蛋白形式的嵌合抗体(IgG型),并分析比较了人q1区对于改造后的工程抗体生物活性的影响。 1.抗CD20嵌合抗体TGLA的体内、外生物活性研究免疫共沉淀的结果证实：抗CD20嵌合抗体TGLA可特异性的沉淀出CD20蛋白；并且间接荧光分析的结果进一步表明TGLA可与CD20+细胞系特异性结合。补体依赖的细胞毒性作用(complement-dependent cytotoxicity, CDC)和抗体依赖性细胞介导的细胞毒性作用(antibody-dependent cell-mediated cytotoxicity, ADCC)分析证明：TGLA能够特异性地杀伤恶性B淋巴细胞；应用TGLA治疗荷瘤裸鼠,平均存活期延长了1.7倍。 2.抗CD20抗体1-28的结构模拟、改造及结合活性检测在计算机模拟基础上,把鼠源抗CD20抗体1-28(IgM)的轻、重链可变区基因和人IgG1的Fc或完全的重链恒定区融合,分别构建了两种工程抗体LH23(VL-Linker-VH-Hγ-Cγ2-Cγ3)和LH1-3 (VL-linker-VH-Cγ1-Hγ-Cγ2-Cγ3)。把构建的表达载体瞬时转染293T细胞后,通过western blot证实两种工程抗体均可被抗人IgG抗体所识别；FACS分析证实两种工程抗体都可与CD20+细胞系特异性结合,其中LH1-3与CD20+细胞系Daudi、Raji细胞结合活性明显高于LH23,与同源模建、分子力学方法预测的结果一致。这些实验结果充分说明人q1区的刚性结构对于由IgM型改造成的IgG型抗体是十分关键的。
[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 纬 -C 纬 2-C 纬 3) and LH1-3 VL-linker-VH-C 纬 -H 纬 -C 纬 -C 纬 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 纬 -C 纬 2-C 纬 3 and LH1-3 mAb VL-linker-VH-C 纬 1-H 纬 -C 纬 -C 纬 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.
【学位授予单位】：河北大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【引证文献】