GENERATION OF HUMAN ANTIBODIES SPECIFIC FOR HUMAN CELL SURFACE ANTIGENS BY GENETIC ENGINEERING

通过基因工程产生针对人类细胞表面抗原的人类抗体

基本信息

批准号：
07557318
负责人：
KUMAGAI Izumi
金额：
$ 2.82万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

It has been known that various biomolecules displayd on cell surfaces characterize the eukaryote cells. Human monoclonal antibody specific for these biomolecules on human cell surfaces have immense potentials for medical supplies, e.g.regulation of cell function and examination of diseases. However, the difficulty in preparation of hybridoma producing a specific antibody have restricted the supply of the antibody molecules. In this research, human red cell is taken up as a model cell, and two goals have been focused ; (1) preparation of human antibody molecules specific for human cell surface antigen by further improvement of phage display system developed recently, and construction of systematical preparation system of human antibody fragments ; (2) grafting other functions and/or multivalency into an antibody fragment prepared by genetic engineering, and its application of various inspection of cell diseases. The results can be summarized as follows ; (1) An expression system for sin … More gle-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli. The scFv fragment expressed by the bacteria produced specific agglutination of human D positive red cells in the presence of an anti-peptide tag antibody. (2) In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, becterial alkaline phosphatase (BAP). The fusion protein produced significant agglutination of human red blood cells with D antigen, confirming that the bacterially expressed fusion protein is a functional bivalent antibody fragment. Specific agglutination of D positive red cells by D10 scFv-BAP was enhanced in the presence of anti-BAP antibody, suggesting that further multimerization of scFv led to highly efficient cell agglutination. By grafting BAP enzymatic activity into the scFv fragment (enzyme-linked scFv), blood typing could conveniently be performed. These results indicate that bacterially expressed scFv and scFv-BAP would be of practical use in blood typing. Less

众所周知，细胞表面展示的各种生物分子表征了人类细胞表面上这些生物分子的人单克隆抗体在细胞功能调节和疾病检查等医疗用品方面具有巨大的潜力。本研究以人红细胞为模型细胞，重点关注两个目标：（1）制备针对人细胞表面的人抗体分子。抗原通过对最近开发的噬菌体展示系统的进一步改进，构建系统化的人源抗体片段制备体系；(2)将其他功能和/或多价嫁接到基因工程制备的抗体片段上，及其在细胞疾病的各种检查中的应用。结果可概括如下：（1）在大肠杆菌中建立了Rh血型系统中抗D抗原的sin gle链Fv片段（scFv）的表达系统。在抗肽标签抗体存在下，细菌对人 D 阳性红细胞产生特异性凝集 (2) 为了构建用于直接细胞凝集的二价 D10 scFv，将 scFv 与二聚蛋白细菌融合。碱性磷酸酶（BAP）使人红细胞与 D 抗原发生显着凝集，证实细菌表达的融合蛋白是功能性二价抗体。在抗 BAP 抗体存在的情况下，D10 scFv-BAP 对 D 阳性红细胞的特异性凝集作用增强，这表明通过将 BAP 酶活性移植到 scFv 片段中，scFv 的进一步多聚化可实现高效的细胞凝集。连接的scFv)，可以方便地进行血型定型。这些结果表明细菌表达的scFv和scFv-BAP在血型定型中具有实际用途。