微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞

目的 探讨抗P-糖蛋白（P-gP）／抗CD3微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞的作用。方法 采用抗E-tag亲和层析柱分离纯化抗P-gp／抗CD3微型双功能抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定;采用^51Cr释放实验,测定抗P-gP／抗CD3微型双功能抗体介导的人T细胞体外靶向杀伤活性;采用多药耐药（MDR）细胞系KB／MDR、敏感KB细胞裸鼠移植瘤模型,测定该微型双功能抗体介导的体内靶向杀伤活性。结果 纯化的抗P-gp／抗CD3微型双功能抗体,在相对分子质量28000和26000处各有1条蛋白带。在抗P-gp／抗CD3微型双功能抗体存在的情况下,激活的T淋巴细胞能够裂解KB／MDR细胞,且随着效靶比的增高而增高。抗P-gp／抗CD3微型双功能抗体联合人T淋巴细胞能有效抑制KB／MDR细胞裸鼠移植瘤的生长,而对敏感KB细胞移植瘤的生长无抑制作用。结论 抗P-gP／抗CD3微型双功能抗体在体内外均能介导人T淋巴细胞杀伤表达P-gP抗原的KB／MDR细胞,是有望用于耐药实体瘤临床治疗的双特异性抗体。

An anti-P-gp/anti-CD3 bispecific antibody cytotoxic to human multidrug resistant KB cells

OBJECTIVE: To study the specific cytotoxicity mediated by anti-P-gp/anti-CD(3) diabodies in multidrug resistant solid tumor using P-gp as target. METHODS: The anti-P-gp/anti-CD(3) diabodies were secreted from E. coli strain 16C9 containing the expression plasmid PAYZDCP, grown at 30 degrees C in a shaker flask; the diabodies were purified by affinity chromatography and identified by SDS-PAGE; the effect of the anti-P-gp/anti-CD(3) diabody mediated lysis of P-gp-expressing tumor cells was assayed by (51)Cr release assay in vitro, and by human KB nude mouse xenograft models in vivo. RESULTS: The diabodies were generated by bacteria as a soluble functional form and purified by one-step affinity chromatography with a yield > 4 mg/L culture medium. In (51)Cr release assay, the diabodies targeted human activated T cells to lyse P-gp(+)-KB/MDR cells in a dose-dependent manner. It suggested that the diabody was able to induce an efficient lysis of the target cells by human T cells in vitro. When combined with activated human T cells, the diabody significantly inhibited the growth of KB/MDR, but had no effect on KB xenografts. CONCLUSION: The anti-P-gp/anti-CD(3) bispecific antibody is a potent agent for targeting human T lymphocytes to lyse solid tumor cells overexpressing P-gp in vitro and in vivo.