Development Of Quantitative Förster Resonance Energy Transfer (QFRET) Based High Throughput (HTS) Screening For PD-1/PD-L1 Immune-Checkpoint Assay

Abstract

Author(s): Xaypraseuth, Amanda; Madahar, Vipul; Liao, Jiayu | Abstract: Programmed cell death protein 1 (PD-1) and programmed cell death 1 - ligand 1 (PD-L1) are immune-checkpoint proteins that play an important part in cancer immunity. PD-1 is a protein on the surface of cells that down-regulates the immune system1 while PD-L1 is a protein on some normal and cancer cells. The interaction of these proteins play a major role in tumor immune escape, inhibiting T lymphocyte proliferation and survival functions. To combat this issue, targeting these immune checkpoint proteins with monoclonal antibodies (mAbs) has become the turning point in cancer treatment. However, limitations were found using mAbs such as the cost of administration, its high molecular weight, and its lack of clinical efficacy. Recently, researchers are investigating small molecule inhibitors to target the PD-1/PD-L1 mechanism instead. With CA-170 as the only small-molecule modulator in clinical trials targeting PD-1, it is essential to research options that can contribute to cancer treatments. This study provides a novel, rapid assessment for PD-1/PD-L1 interaction with the use of FRET- based kinetic analysis. PD-1/PD-L1 binding will be quantified by fluorescence using donor and acceptor pairs, CyPet and Ypet, which were bound to PD-L1 and PD-1, respectively. From this study, we calculated a Kd value of 0.31±0.13 and developed an HTS assay with a Z’ value g 0.7, values that validate the robustness and efficacy of this assay. With the development of this type of screening, it will be easy to contribute to small molecule inhibitor discovery and the growing field of cancer immunotherapy.