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. Graphical Abstract

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