Establishment of Human PD-1/PD-L1 Blockade Assay Based on Surface Plasmon Resonance (SPR) Biosensor

Abstract

Blockade of the programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) axis is a promising strategy for cancer immunotherapy. Although antibody-based PD-1/PD-L1 inhibitors have shown remarkable results in clinical cancer studies, their inherent limitations underscore the significance of developing novel PD-1/PD-L1 inhibitors. Small molecule inhibitors have several advantages over antibody-based inhibitors, including favorable tumor penetration and oral bioavailability, fewer side effects, easier administration, preferred biological half-life, and lower cost. However, small molecule inhibitors that directly target the PD-1/PD-L1 interaction are still in the early development stage, partially due to the lack of reliable biophysical assays. Herein, we present a novel PD-1/PD-L1 blockade assay using a surface plasmon resonance (SPR)-based technique. This blockade assay immobilizes human PD-1 on a sensor chip, which interacts with PD-L1 inhibitors or negative PD-L1 binders with human PD-L1 protein at a range of molecular ratios. The binding kinetics of PD-L1 to PD-1 and the blockade rates of small molecules were determined. Compared to other techniques such as PD-1/PD-L1 pair enzyme-linked immunosorbent assay (ELISA) and AlphaLISA immunoassays, our SPR-based method offers real-time and label-free detection with advantages including shorter experimental runs and smaller sample quantity requirements.Key features• A SPR protocol screens compounds for their capacity to block the PD-1/PD-L1 interaction.• Validation of PD-1/PD-L1 interaction, followed by assessing blockade effects with known inhibitors BMS-1166 and BMS-202, and a negative control NO-Losartan A.• Analysis of percentage blockade of PD-1/PD-L1 of the samples to obtain the IC50.• Broad applications in the discovery of small molecule–based PD-1/PD-L1 inhibitors for cancer immunotherapy.Graphical overview