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

P-糖蛋白与趋化因子单链抗体融合蛋白的制备及应用研究

• 批准号: 13557053
• 负责人: SONE Saburo
• 金额: $ 6.27万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557053/
• 关键词: Multi-drug resistance; P-glycoprotein; MRK16; single chain antibody; P糖蛋白; single chan抗体; ケモカイン; 薬剤耐性; 抗体治療; 免疫療法; MRK-16; MCP-1; Multi-drug resistance; P-glycoprotein; MRK16; single chain antibody; P糖蛋白; single chan抗体; ケモカイン; 薬剤耐性; 抗体治療; 免疫療法; MRK-16; MCP-1

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.

MDR1基因产物P-糖蛋白在癌细胞的多药耐药性中起关键作用。 P-糖蛋白是能量依赖性泵，可挤出各种化学治疗药物。 P-糖蛋白的过表达导致体外和癌症患者的肿瘤细胞系的多药耐药性。因此，已经将P-糖蛋白作为癌症治疗的分子靶点引起了很多关注。 MRK16是抗P-糖蛋白的单克隆抗体，并与P-糖蛋白的外部结构域结合。它部分抑制了多药耐药细胞中某些化学治疗药物的外流，并导致体内肿瘤回归。由于在人类临床环境中使用完整的鼠抗体的局限性，我们设计了MRK16的单链抗体片段（SCFVMRK16）。克隆MRK16的可变区域后，将重链和轻链碎片与接头融合，并从大肠杆菌中产生重组蛋白。尽管与MRK16相比，SCFVMRK16与P-糖蛋白的结合活性少200-300倍，但我们通过流式细胞仪分析证实，它确实识别了P-糖蛋白。此外，SCFVMRK16可以抑制MRK16的活性结合P-糖蛋白。这些结果表明SCFVMRK16的治疗潜力。