抗CTLA-4嵌合抗体的制备及生物学活性鉴定

Preparation and characterization of a chimeric anti-human CTLA-4 monoclonal antibody

制备新型抗CTLA-4人鼠嵌合抗体并进行活性鉴定。通过杂交瘤技术获得高亲和力小鼠抗人CTLA-4单克隆抗体22G11和16C11;利用分子克隆技术将鼠源抗体可变区基因与人源抗体恒定区拼接后,最终通过CHO-K1工程株细胞表达高亲和力抗CTLA-4嵌合抗体。经SDS-PAGE电泳显示最终获得了纯度高于90%的CTLA-4嵌合抗体c22G11和c16C11,抗原结合活性结果表明两株嵌合抗体都能很好地与Jurkat细胞结合,竞争抑制实验表明它们都能与各自对应的鼠源抗体竞争。据此,本实验获得了两株抗人CTLA-4胞外区的高亲和力和特异性嵌合抗体。

Through the hybridoma technique, we got two hybridoma cells secreting antiCTLA4 mAb with high affinity, which were named 22Gll and 16Cll respectively. To construct the chimeric antibody light chain and heavy chain expression vectors, the cloned antiCTL4 antibody＇s light chain （VL） and heavy chain variable region cDNAs （VH） were respectively fused to the human antibody light and heavy chain constant region genes （CH）. Then appropriate light and heavy chain expres sion vectors were cotransfected into CHOK1 cells and the clones producing the highest amount of chimeric antibodies were selected. In the end, the purity of the two chimeric antibodies was more than ninety percent. Both of them can bind Jurkat cells specifically as did the commercial antiCTLA4 mAb. Western blotting analysis showed that mAbs 22Gll and 16Cll reacted with one specific band with a molecular weight of about 22 kD, further indicating that 22Gll and 16Cll specifically recognized CTLA4 molecule. Antigenbinding activity assay demonstrated that both of the two mAbs were shown to bind well to Jurkat cells, indicating that the two chimeric antibodies were correctly constructed and produced. In the competitive binding assay, c22Gll and c16Cll could effectively compete with their respective parental mouse antibodies for binding to Jurkat cells. The avidity （mean IC50±SD） of c22Gll and c16Cll was equal to that of 22Gll and 16Cll respectively, which indicated that the chimeric antibodies possessed affinity and specificity similar to those of the original murine antibodies.