| 题名 | CD176 scFv 小分子抗体表达、鉴定及应用 |
| 作者 | 刘江南 |
| 学位类别 | 硕士 |
| 答辩日期 | 2015-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曹毅 |
| 关键词 | CD176 抗原 scFv 小分子抗体 免疫治疗 人源化抗体 |
| 其他题名 | Constuction, identification and application of CD176 scFv |
| 中文摘要 | CD176（Thomsen-Friedenreich抗原）是一种隐性血型糖抗原，是MN血型决定簇的前体物质。在正常细胞中，由于唾液酸的掩盖，CD176抗原并不暴露出来。然而在肿瘤细胞中，其糖基化功能障碍，缺少唾液酸修饰，导致CD176抗原出现在细胞表面，因此CD176抗原可视为一种肿瘤抗原。多种恶性肿瘤细胞表面均表达该抗原，如乳房癌、胃癌、肠癌、肝癌、肾癌、肺癌、淋巴瘤及白血病等，但在正常成人组织中并不表达。有研究证实，肿瘤的发展浸润和转移与CD176抗原息息相关。我们的前期工作也标明，大部分白血病细胞系都高表达CD176抗原，而且用TF抗体处理可以明显诱导其凋亡。通过CD176抗血清对CD176+阳性白血病小鼠的体内治疗结果发现，CD176抗血清可以有效抑制白血病细胞的肝转移，有效诱导体内白血病细胞的凋亡，并且明显延长患病小鼠的生存时间。 为了研发治疗性抗体药物，我们通过aGP免疫小鼠，构建小鼠脾B细胞cDNA文库，然后成功构建了抗CD176抗原的噬菌体展示文库。接着，我们通过ELISA筛选的方式，成功筛选到两株对CD176抗原有强结合能力的菌株 (命名为T34和T48)。这两株抗体有着不同DNA序列，我们成功克隆了T34和T48中的scFv基因，并且构建了基于T48菌株中scFv基因的3个scFv-His tag，scFv-scFv, scFv-AP表达载体，及T34的scFv-His表达载体。 我们成功的将34/48小分子抗体在大肠杆菌BL21中高效表达，通过亲和层析，透析及凝胶层析等一系列的纯化方法，我们成功得到了纯化的34/48-His tag抗体。通过流式细胞术检测了该抗体可以与白血病细胞系KG1有效的结合，并且通过免疫荧光的方法再一次证实这两个小分子抗体可以与CD176阳性的胃癌及肠癌细胞系相结合。最后我们用此抗体进行阻断实验，这两个抗体可以明显抑制CD176阳性的癌细胞对正常肝细胞及血管内皮细胞的粘附作用，因此该小分子抗体在体外可以有效阻止癌细胞的粘附。 为了更好的将此小分子抗体用于癌症的免疫治疗，我们展开了人源化抗体的构建，将scFv小分子通过融合PCR与人IgG1 Fc片段结合在一起，形成scFv-Fc人源化嵌合抗体，将此载体转入哺乳动物细胞CHO-K1及HEK293分别进行真核表达，目前，我们已经成功构建了真核表达载体，并且尝试了胞内表达，验证了其活性。为未来进行大量分泌表达及纯化奠定了基础。 CD176抗原在正常组织中的不表达以及在多数肿瘤中表达的特征使得其成为一种重要的分子靶标。我们的研究也表明，CD176抗体对CD176+白血病有着显著的治疗疗效，因此我们通过噬菌体展示文库筛选的方式，构建高效的CD176小分子抗体的表达载体，并成功的进行了人源化的改造，为以后CD176人源化抗体的制备与应用做前期研究，具有十分重要的科学价值。 |
| 英文摘要 | CD176 （Thomsen-Friedenreich antigen，chemical structure is Gal1-3GalNAc1-R）is a precursor of MN blood type antigenic determinant. In adult human normal and benign tissues, CD176 is masked by terminal sialylation, but it is exposed during tumorigenesis as a tumor-associated antigen. CD176 exposes on many kinds of cancer cells, such as breast cancer cells, colon cancer cells, lung cancer cells and leukemia cells, but not on normal tissue cells. Previous researches demonstrated that CD176 expression has a significant relationship with the infiltration, metastasis and development of cancer. Our previous research showed that CD176 is expressed on many kinds of leukemia cells and CD176 antibody can induce the apoptosis of these kinds of leukemia cells. Basing on the research about CD176 antiserum treatment in a murine model of leukemia, we know that CD176 antibody could inhibit CD176+cancer cell metastasis to bone marrow, spleen, lung and liver through blocking the adhesion of cancer cells to the endothelium and hepatocytes; CD176 antibody could mediate the elimination of CD176+cancer cells through complement dependent cytotoxicity and/or antibody dependent cellular cytotoxicity; and CD176 antibody could induce apoptosis of CD176+ leukemia cells. For development of therapeutic CD176 antibody, we immunized mouse with aGP protein, and successfully constructing the phage display library of anti-CD176 antigen. Then, we have successfully screened two clones (named T34 and T48) with a high affinity to the CD176 antigen through ELISA method. Afterwards, we clone T34/T48 scFv gene and construct three expression vectors, scFv-His tag, scFv-scFv, scFv-AP. We have expressed 34/48 scFv antibody in BL21 and purified the scFv by affinity chromatography, dialysis, HPLC and ion-exchange column chromatography. Furthermore, we detect their affinity to CD176 antigen by flow cytometry and immunofluorescence. The result demonstrate that these scFv antibody can inhibit CD176+ cancer cells adhesion to endothelium cell and hepatocytes. Therefore, 34/48scFv antibody could be used to cancer, biotherapy in the future. Additionally, we construct a humanized antibody which will have some cytotoxicity like ADCC or CDC. We have linked these scFv to human IgG1 Fc part and got a recombinant antibody. This humanized antibody might be used to cancer immunotherapy preferably in the future. CD176 is expressed on the most cancer cells and is not expressed on normal cells，which indicates that CD176 might be a promising biomarker. Our research data also demonstrated that CD176 antibody has a significant therapy effect to the CD176+ leukemia. Therefore, it has the scientific and clinical significance to construct a humanized CD176 antibody so that it could be used to cancer immunotherapy in the future. |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10127]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 刘江南. CD176 scFv 小分子抗体表达、鉴定及应用[D]. 北京. 中国科学院研究生院. 2015. |
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