【摘要】：鼠源單克隆抗體在臨床應(yīng)用中有以下缺點(diǎn):誘發(fā)人體產(chǎn)生人抗小鼠抗體(human anti-mounse antibody,HAMA)反應(yīng),在人體內(nèi)半壽期短,不能夠有效地激活人體的生物效應(yīng)功能,如補(bǔ)體依賴的細(xì)胞毒作用(complement-dependent cytotoxicity,CDC)及抗體依賴性細(xì)胞介導(dǎo)的細(xì)胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)。通過分子生物學(xué)技術(shù)對(duì)鼠抗體進(jìn)行人源化改造在一定程度上可以克服這些缺點(diǎn)。本室制備的鼠源抗CD20單克隆抗體(monoclonal antibody,mAb)1-28具有潛在的應(yīng)用價(jià)值。本研究在對(duì)mAb 1-28(簡稱1-28)功能進(jìn)行驗(yàn)證的基礎(chǔ)上,通過計(jì)算機(jī)輔助分子設(shè)計(jì)和分子生物學(xué)技術(shù)對(duì)其進(jìn)行改造,構(gòu)建了抗CD20嵌合抗體C1-28和新型小分子抗CD20抗體5S(ScFv-Fc),并對(duì)其抗原結(jié)合特性及通過CDC殺傷腫瘤細(xì)胞的功能進(jìn)行了初步研究。 1 抗CD20單克隆抗體1-28的免疫學(xué)特性及功能分析 首先分析了1-28與靶細(xì)胞的結(jié)合活性。間接免疫熒光的結(jié)果顯示,1-28可與人B細(xì)胞淋巴瘤Daudi細(xì)胞和Raji細(xì)胞結(jié)合,而不與人T細(xì)胞系Jurkat細(xì)胞結(jié)合,表明1-28與靶細(xì)胞的結(jié)合是特異的。流式細(xì)胞術(shù)分析結(jié)果顯示,1-28與Daudi和Raji細(xì)胞結(jié)合的陽性率達(dá)99%以上。相對(duì)親和力(relative binding affinity)測試結(jié)果顯示,1-28的親和力為美羅華親和力的1/12左右。通過ELISA法測定出1-28的親和常數(shù)為K=1.6×10~(10) M~(-1)。競爭實(shí)驗(yàn)的結(jié)果提示1-28與另外兩種抗CD20抗體(美羅華、2H7)識(shí)別的是不同的抗原表位。1-28對(duì)Raji細(xì)胞和Daudi細(xì)胞都具有生長抑制效應(yīng),但是程度不同。另外,1-28可以誘導(dǎo)Raji細(xì)胞和Daudi細(xì)胞發(fā)生凋亡。1-28還可通過激活補(bǔ)體殺傷靶細(xì)胞。以Raji細(xì)胞為研究對(duì)象時(shí),其半數(shù)殺傷濃度為1.26 nmol/L。1-28對(duì)非靶細(xì)胞Jurkat沒有CDC的功能,提示這種殺傷是特異的。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 耿樹生;抗CD20抗體的改造及其生物活性研究[D];河北大學(xué);2007年

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本文編號(hào)：2461252