【作者】 王玉刚；

【导师】 沈倍奋；

【作者基本信息】 中国人民解放军军事医学科学院 ， 分子免疫学， 2005， 博士

【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=1.6×10¹⁰ 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。更多还原

【Abstract】 The clinical application of murine monoclonal antibodies is associated with a number of drawbacks, including inducement of human anti-mouse antibody （HAMA）, short circulating time in vivo, and the inability to fully harness the effectors （ADCC） and complement （CDC） repertoire of the human immune system. Genetic engineering has been used to humanize mouse mAb and can overcome the drawbacks significantly. Our group developed a mouse anti-human CD20 monoclonal antibody 1-28, which possessed potential clinical values. The immunological characteristics of mAb 1-28 were tested. With computer-aided molecular modeling design method and DNA recombination technology, chimeric anti-CD20 antibody and single-chain Fv （scFv） with human lgG1 hinge and Fc regions, designated 5S, were produced. Their binding activity and complement-dependent cytotoxicity （CDC） were analyzed. 1 The immunological characters study on mAb 1-28Specific binding activity of mAb 1-28 was verified by flow cytometry method. The relative binding affinity of mAb 1-28 was only about 1/12th of Rituxan. K value of 1-28 was 1.6×10¹⁰M^-1 measured by Scatchard analysis. The results of competitive binding assay showed that both 2H7 （mouse anti-human CD20 antibody） and Rituxan （chimeric anti-CD20 antibody） could not inhibit the binding of 1-28 to CD20 antigen expressed on the surface of Raji cells. 1-28 could alone inhibit CD20+ cells’ proliferation and was sufficient to induce apoptosis of Raji cells and Daudi cells. 1-28 could also fix and activate complement to kill CD20+ cells, such as Raji, while it’s ineffective to Jurkat cells.2 Expression and characterization of Chimeric Anti-CD20 AntibodyThe light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR method, and then cloned to T vector, sequenced. MAb 1-28 protein was separated by reduced SDS-PAGE, and the light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. The light- and heavy-chain DNA sequences were verified by their protein sequences. To acquire a positive control in the experiments, V genes of 2H7 were synthesized by overlapping PCR method. Both V genes of 1-28 and 2H7 were cloned into chimeric antibody expression vector （pCMV-VH & pCMV-VL）, generating chimeric anti-human CD20 mAb expression vectors. Plasmids were transfected into 293T cells with Lipofectamine? 2000. High level expression of the two anti-human CD20 chimeric mAbs were obtained and their m.w. in agreement with that of human IgG. The binding activity of C2H7 was well reserved, but C1-28 lost its binding activity to Daudi and Raji cells completely. In complement-dependent cytotoxicity assay, C2H7 could fix and activate both human complement and rabbit complement to kill target cells specifically.3 Design of a new kind of anti-CD20 antibodyThe spatial structure of mAb 1-28 variable region was modeled theoretically with homology method carried on http://www.expasy.ch. The stability of mAb 1-28 Fv was analyzed according to inter-molecular hydrogen-bond forming theory and reaction free energy theory. The result showed that the stability of the complex Fv was weak due to the structural non-match and electrostatic non-complementarity. The weak stability of the complex Fv induced the low bioactivity. The residues characters of N and C terminal of antibody 1-28 were studied based on distance geometry and computer graphics technology. The single chain Fv antibody （abbr. scFv） was constructed and the mode was VH-Linker-VL. The result showed that the stability of the antibody 1-28 was increased while the active domain conformation was not changed due to the更多还原