PD-1/PD-L1轴阻断是癌症免疫治疗的一种有希望的策略。虽然基于抗体的PD-1/PD-L1抑制剂在临床癌症研究中显示出明显效果，但其固有局限性强调了开发新型PD-1/PD-L1抑制剂的重要性。与基于抗体的抑制剂相比，小分子抑制剂具有几个优点，包括有利的肿瘤渗透性和口服生物利用度，副作用较少，给药更容易，生物半衰期更好，成本更低。然而，直接靶向PD-1/PD-L1相互作用的小分子抑制剂仍处于早期开发阶段，部分原因是缺乏可靠的生物物理测定方法。在此，我们提出了一种基于表面等离子共振（SPR）的新型PD-1/PD-L1阻断测定方法。该阻断测定方法将人源PD-1固定在传感器芯片上，与PD-L1抑制剂或负向PD-L1结合物在一系列分子比例下相互作用。确定了PD-L1与PD-1的结合动力学以及小分子抑制剂的阻断速率。与其他技术（如PD-1/PD-L1配对酶联免疫吸附试验（ELISA）和AlphaLISA免疫测定）相比，我们的SPR方法提供了实时和无标记检测，具有实验运行时间更短和样本数量要求更少的优势。关键特点是SPR协议用于筛选化合物对PD-1/PD-L1相互作用的阻断能力。通过验证PD-1/PD-L1相互作用，再利用已知的抑制剂BMS-1166和BMS-202以及阴性对照物质NO-Losartan评估阻断效果。分析样品对PD-1/PD-L1阻断的百分比来获得IC50。在小分子PD-1/PD-L1抑制剂的癌症免疫治疗的发现中具有广泛应用。图形概述。©版权：© 2023年作者；本文采用CC BY许可下的开放获取文章。

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.©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.