Namai Fu, Sumiya Shunsuke, Nomura Natsumi, Sato Takashi, Shimosato Takeshi
Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano, 399-4598, Japan.
AMB Express. 2023 Jan 12;13(1):4. doi: 10.1186/s13568-023-01509-y.
Here, we developed a genetically modified lactic acid bacteria (gmLAB) that produces green fluorescent protein (GFP)-conjugating, anti-programmed death-ligand 1 (PD-L1) single-chain variable fragments (scFv) for use as an anti-cancer device that targets immune checkpoint molecules. Since PD-L1 plays a key role as an immune checkpoint molecule in the tumor microenvironment, inhibition and detection of PD-L1 are important in cancer research. The anti-PD-L1 scFv was designed based on atezolizumab, a humanized IgG1 monoclonal antibody, and integrated into a lactococcal GFP gene expression vector. Gene expression from the constructed gmLAB was confirmed by western blotting and GFP fluorescence. The ability of GFP-conjugating anti-PD-L1 scFv against the target antigen, PD-L1 protein, was shown using an enzyme-linked immunosorbent assay. Finally, the ability to recognize PD-L1-expressing tumor-cell lines was confirmed using flow cytometry and fluorescence microscopy. Our results suggest that the gmLAB could be applied to in vivo imaging in cancer as an affordable diagnostic/treatment tool.
在此，我们开发了一种基因工程改造的乳酸菌（gmLAB），它能产生与绿色荧光蛋白（GFP）结合的抗程序性死亡配体1（PD-L1）单链可变片段（scFv），用作靶向免疫检查点分子的抗癌装置。由于PD-L1在肿瘤微环境中作为免疫检查点分子发挥关键作用，因此PD-L1的抑制和检测在癌症研究中很重要。抗PD-L1 scFv是基于人源化IgG1单克隆抗体阿替利珠单抗设计的，并整合到乳酸球菌GFP基因表达载体中。通过蛋白质免疫印迹法和GFP荧光证实了构建的gmLAB的基因表达。使用酶联免疫吸附测定法展示了与GFP结合的抗PD-L1 scFv对靶抗原PD-L1蛋白的结合能力。最后，使用流式细胞术和荧光显微镜确认了识别表达PD-L1的肿瘤细胞系的能力。我们的结果表明，gmLAB作为一种经济实惠的诊断/治疗工具，可应用于癌症的体内成像。
