基于计算机辅助分子设计的抗CD20单克隆抗体的改造
发布时间：2019-04-19 20:24
【摘要】：鼠源单克隆抗体在临床应用中有以下缺点:诱发人体产生人抗小鼠抗体(human anti-mounse antibody,HAMA)反应,在人体内半寿期短,不能够有效地激活人体的生物效应功能,如补体依赖的细胞毒作用(complement-dependent cytotoxicity,CDC)及抗体依赖性细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)。通过分子生物学技术对鼠抗体进行人源化改造在一定程度上可以克服这些缺点。本室制备的鼠源抗CD20单克隆抗体(monoclonal antibody,mAb)1-28具有潜在的应用价值。本研究在对mAb 1-28(简称1-28)功能进行验证的基础上,通过计算机辅助分子设计和分子生物学技术对其进行改造,构建了抗CD20嵌合抗体C1-28和新型小分子抗CD20抗体5S(ScFv-Fc),并对其抗原结合特性及通过CDC杀伤肿瘤细胞的功能进行了初步研究。 1 抗CD20单克隆抗体1-28的免疫学特性及功能分析 首先分析了1-28与靶细胞的结合活性。间接免疫荧光的结果显示,1-28可与人B细胞淋巴瘤Daudi细胞和Raji细胞结合,而不与人T细胞系Jurkat细胞结合,表明1-28与靶细胞的结合是特异的。流式细胞术分析结果显示,1-28与Daudi和Raji细胞结合的阳性率达99%以上。相对亲和力(relative binding affinity)测试结果显示,1-28的亲和力为美罗华亲和力的1/12左右。通过ELISA法测定出1-28的亲和常数为K=1.6×10~(10) M~(-1)。竞争实验的结果提示1-28与另外两种抗CD20抗体(美罗华、2H7)识别的是不同的抗原表位。1-28对Raji细胞和Daudi细胞都具有生长抑制效应,但是程度不同。另外,1-28可以诱导Raji细胞和Daudi细胞发生凋亡。1-28还可通过激活补体杀伤靶细胞。以Raji细胞为研究对象时,其半数杀伤浓度为1.26 nmol/L。1-28对非靶细胞Jurkat没有CDC的功能,提示这种杀伤是特异的。
[Abstract]:Mouse monoclonal antibody has the following disadvantages in clinical application: it can induce human anti-mouse antibody (human anti-mounse antibody,HAMA) reaction, and its half-life is short in human body, so it can not effectively activate the biological effect function of human body. Such as complement dependent cytotoxicity (complement-dependent cytotoxicity,CDC) and antibody dependent cell mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity,ADCC). Humanization of mouse antibodies by molecular biological techniques can overcome these shortcomings to some extent. The mouse anti-CD20 monoclonal antibody (monoclonal antibody,mAb) 1 / 28 prepared in our laboratory has potential application value. In this study, on the basis of the verification of the function of mAb 1x28, it was modified by computer-aided molecular design and molecular biology technology. The anti-CD20 chimeric antibody C1A28 and the novel small-molecule anti-CD20 antibody 5S (ScFv-Fc) were constructed, and their antigen binding properties and killing ability of tumor cells through CDC were studied. 1Immunologic characteristics and functional analysis of anti-CD20 monoclonal antibody 1? 28? first, the binding activity of 1? 28 to target cells was analyzed. The results of indirect immunofluorescence showed that 1 ~ (28) could bind to Daudi cells and Raji cells of human B-cell lymphoma, but not to Jurkat cells of human T cell line, indicating that the binding of 1 ~ (- 28) to target cells was specific. The results of flow cytometry showed that the positive rate of binding to Daudi and Raji cells was more than 99%. The results of relative affinity (relative binding affinity) showed that the affinity of 1 脳 28 was about 1 / 12 of that of melohua. The affinity constant of 1 脳 10 ~ (10) M ~ (- 1) was determined to be 1.6 脳 10 ~ (10) M ~ (- 1) by ELISA. The results of the competition test suggested that 1) the antigenic epitopes recognized by the two other anti-CD20 antibodies (2H7) were different. 1. The growth inhibition of Raji cells and Daudi cells was observed in different degrees. 1. The growth inhibition of Daudi cells and Raji cells was found to be different from that of the other two antibodies (MLC, MCAB). In addition, 1-28 could induce apoptosis in Raji cells and Daudi cells. 1-28 could also kill target cells by activating complement. The 50% killing concentration of Raji cells was 1.26 nmol/L.1-28, which did not have the function of CDC on non-target Jurkat cells, suggesting that the killing was specific.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392
本文编号：2461252
[Abstract]:Mouse monoclonal antibody has the following disadvantages in clinical application: it can induce human anti-mouse antibody (human anti-mounse antibody,HAMA) reaction, and its half-life is short in human body, so it can not effectively activate the biological effect function of human body. Such as complement dependent cytotoxicity (complement-dependent cytotoxicity,CDC) and antibody dependent cell mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity,ADCC). Humanization of mouse antibodies by molecular biological techniques can overcome these shortcomings to some extent. The mouse anti-CD20 monoclonal antibody (monoclonal antibody,mAb) 1 / 28 prepared in our laboratory has potential application value. In this study, on the basis of the verification of the function of mAb 1x28, it was modified by computer-aided molecular design and molecular biology technology. The anti-CD20 chimeric antibody C1A28 and the novel small-molecule anti-CD20 antibody 5S (ScFv-Fc) were constructed, and their antigen binding properties and killing ability of tumor cells through CDC were studied. 1Immunologic characteristics and functional analysis of anti-CD20 monoclonal antibody 1? 28? first, the binding activity of 1? 28 to target cells was analyzed. The results of indirect immunofluorescence showed that 1 ~ (28) could bind to Daudi cells and Raji cells of human B-cell lymphoma, but not to Jurkat cells of human T cell line, indicating that the binding of 1 ~ (- 28) to target cells was specific. The results of flow cytometry showed that the positive rate of binding to Daudi and Raji cells was more than 99%. The results of relative affinity (relative binding affinity) showed that the affinity of 1 脳 28 was about 1 / 12 of that of melohua. The affinity constant of 1 脳 10 ~ (10) M ~ (- 1) was determined to be 1.6 脳 10 ~ (10) M ~ (- 1) by ELISA. The results of the competition test suggested that 1) the antigenic epitopes recognized by the two other anti-CD20 antibodies (2H7) were different. 1. The growth inhibition of Raji cells and Daudi cells was observed in different degrees. 1. The growth inhibition of Daudi cells and Raji cells was found to be different from that of the other two antibodies (MLC, MCAB). In addition, 1-28 could induce apoptosis in Raji cells and Daudi cells. 1-28 could also kill target cells by activating complement. The 50% killing concentration of Raji cells was 1.26 nmol/L.1-28, which did not have the function of CDC on non-target Jurkat cells, suggesting that the killing was specific.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392
【引证文献】
相关博士学位论文 前1条
1 耿树生;抗CD20抗体的改造及其生物活性研究[D];河北大学;2007年
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