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

Abstract

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 IC₅₀ 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.