Human CD274 Stable Cell Line - CT26

Colorectal cancer (CRC) is the third leading cause of cancer death worldwide. The prognosis and overall survival of CRC are closely related to the overexpression of programmed death ligand-1 (PD-L1 or CD274). Given that combined therapy can significantly improve the efficacy, researchers constructed doxorubicin (DOX) conjugated and anti-PD-L1 targeting gold nanoparticles (PD-L1-AuNP-DOX) for targeted chemo-photothermal therapy of CRC. DOX and anti-PD-L1 antibody were coupled to the α-terminus of lipoic acid polyethylene glycol-N-hydroxysuccinimide (LA-PEG-NHS) via amide bonds, and LA-PEG-DOX, LA-PEG-PD-L1, and short PEG chains on the surface of gold nanoparticles were linked via thiol-gold covalent bonds to construct PD-L1-AuNP-DOX. The physicochemical characterization and biological studies of PD-L1-AuNP-DOX were carried out under near-infrared (NIR) irradiation. PD-L1-AuNP-DOX was successfully constructed and promoted the effective intracellular uptake of DOX, which showed a significant apoptotic effect in CT-26 cells (66.0%). PD-L1-AuNP-DOX combined with NIR irradiation significantly and synergistically inhibited the proliferation of CT-26 cells in vitro, thereby promoting cell apoptosis and cell cycle arrest. The study showed that PD-L1-AuNP-DOX combined with synergistic targeted chemophotothermal therapy has great potential in the treatment of localized colorectal cancer.

To evaluate the retention of PD-L1 immunoreactivity after PEGylation, different ratios of LA-PEG-NHS to anti-PD-L1 antibody (5:1, 25:1, and 50:1) were prepared and their binding to PD-L1 (CD274) overexpressing CT-26 cells was assessed using flow cytometry. Flow cytometric analysis of LA-PEG-PD-L1 (Figure 1A) showed that the binding rate increased when the molar ratio of LA-PEG-NHS/anti-PD-L1 was decreased. When the anti-PD-L1 antibody was reacted with a 50-fold molar excess of LA-PEG-NHS to prepare the LA-PEG-PD-L1 conjugate, the immunoreactivity of the anti-PD-L1 antibody was only 50%. Flow cytometric analysis of the LA-PEG-PD-L1 immunoconjugate showed that the PD-L1 binding affinity was >90% when the anti-PD-L1 antibody was reacted with a 5-fold molar excess of LA-PEG-NHS. CLSM confirmed that when a 50-fold molar excess of LA-PEG-NHS reacted with the anti-PD-L1 antibody, the cell binding rate decreased (Figure 1B). On the other hand, when the molar ratio of LA-PEG-NHS to anti-PD-L1 of the anti-PD-L1 antibody was 5:1 or 25:1, the cell binding rate was satisfactory. Therefore, a molar ratio of 5:1 was used in subsequent experiments.

Figure 1. Immunoreactivity of LA-PEG-PD-L1. (Emami F, et al., 2019)