Studies on the generation and application of fusion protein of single chain antibody for P-glycoprotein and chemokine

MDR1基因产物p -糖蛋白在癌细胞多药耐药过程中起关键作用。p -糖蛋白是一种能量依赖型泵，可挤出多种化疗药物。p -糖蛋白的过表达导致肿瘤细胞系和肿瘤患者的多药耐药。因此，p糖蛋白作为肿瘤治疗的分子靶点受到了广泛的关注。MRK16是针对p -糖蛋白的单克隆抗体，与p -糖蛋白的外部结构域结合。它部分抑制某些化疗药物在多药耐药细胞中的外排，导致体内肿瘤消退。由于在人类临床环境中使用完整的小鼠抗体的局限性，我们设计了MRK16的单链抗体片段（scFvMRK16）。将MRK16可变区克隆后，将重链和轻链片段与连接子融合，从大肠杆菌中获得重组蛋白。虽然scFvMRK16与p -糖蛋白的结合活性比MRK16低200-300倍，但我们通过流式细胞分析证实它确实识别p -糖蛋白。scFvMRK16可以抑制MRK16结合p -糖蛋白的活性。这些结果表明了scFvMRK16的治疗潜力。

The MDR1 gene product P-glycoprotein plays a key role in multidrug resistance of cancer cells. P-glycoprotein is the energy-dependent pump that extrudes a variety of chemotherapeutic drugs. Overexpression of P-glycoprotein results in multidrug resistance of tumor cell lines in vitro as well as in cancer patients. Therefore, much attention has been paid to P-glycoprotein as a molecular target for cancer treatment. MRK16 is the monoclonal antibody against P-glycoprotein and binds to an external domain of P-glycoprotein. It partially inhibits the efflux of certain chemotherapeutic drugs in multidrug-resistant cells, and leads the tumor regression in vivo. Due to the limitations of using intact murine antibodies in human clinical settings, we engineered a single-chain antibody fragment of MRK16 (scFvMRK16). After cloning the variable region of MRK16, both heavy and light chain fragments were fused with linker, and recombinant protein were produced from Escherichia coli. Although the binding activity of scFvMRK16 to P-glycoprotein was 200-300 fold less compared to MRK16, we confirmed by flow cytometric analysis that it does recognize the P-glycoprotein. Furthermore, scFvMRK16 could inhibit the activity of MRK16 to bind P-glycoprotein. These results indicated the therapeutic potential of scFvMRK16.