Discovery of PD-L1 Peptide Inhibitors from Ascidian Enzymatic Hydrolysates by Affinity Ultrafiltration Coupled to NanoLC-MS/MS.

Anti-PD-1 and anti-PD-L1 antibodies have achieved great clinical success in cancer immunotherapy, and peptide and small molecule inhibitors of PD-1/PD-L1 binding also attract much attention. Ascidians are not only seafood, but are also an important source of bioactive substances, including anti-tumor components. In this study, ascidian enzymatic hydrolysates were found to contain PD-1/PD-L1 inhibitory components. Affinity ultrafiltration (AUF) coupled with the nanoLC-MS/MS method was first applied in screening for PD-L1 peptide inhibitors from ascidian enzymatic hydrolysates. Two anti-PD-L1 ascidian peptides, C5 (LDVVIHTVTYGDR) and S2 (VLRDNIQGITKPAIR), were filtered out from the ascidians Ciona intestinalis and Styela clava, respectively. C5 and S2 showed moderate anti-PD-1/PD-L1 effects with the IC(50) values of 33.9 µM (C5) and 112.8 μM (S2), respectively, by homogenous time-resolved fluorescence (HTRF) binding assay, and the K(D) values of 22.9 µM (C5) and 29.1 µM (S2), respectively, by surface plasmon resonance (SPR) assay. The results of this study suggest that ascidian enzymatic hydrolysates may be a potential source of bioactive peptides with anti-PD-1/PD-L1 activity.