An Imaging-Based Assay to Measure the Location of PD-1 at the Immune Synapse for Testing the Binding Efficacy of Anti-PD-1 and Anti-PD-L1 Antibodies

PD-1 is an immune checkpoint on T cells. Antibodies to PD-1 or its ligand PD-L1 are gaining popularity as a leading immunotherapy approach. In the US, 40% of all cancer patients will be treated with anti-PD-1 or anti-PD-L1 antibodies but, unfortunately, only 30% will respond, and many will develop immune-related adverse events. There are nine FDA-approved anti-PD-1/PD-L1 antibodies, and approximately 100 are in different stages of clinical development. It is a clinical challenge to choose the correct antibody for a given patient, and this is critical in advanced malignancies, which often do not permit a second-line intervention. To resolve that, an in vitro assay to compare the performance of the different anti-PD-1/PD-L1 antibodies is not only a critical tool for research purposes but also a possible tool for personalized medicine. There are some assays describing the binding affinity and function of anti-PD-1/PD-L1 antibodies. However, a significant limitation of existing assays is that they need to consider the location of PD-1 in the immune synapse, the interface between the T cell and tumor cells, and, therefore, ignore a critical component in its biology. To address this, we developed and validated an imaging-based assay to quantify and compare the ability of different anti-PD-1/PD-L1 antibodies to remove PD-1 from the immune synapse. We correlated that with the same antibodies' ability to increase cytokine secretion from the targeted cells. The strong correlation between PD-1 location and its function in vitro and in vivo within the antibody treatment setting validates this assay's usability, which is easily recordable and straightforward. Key features • Live-cell imaging quantifies and compares how anti-PD-1 and anti-PD-L1 antibodies disrupt PD-1 localization, causing the removal of PD-1 during immune synapse formation. • Hao et al. [1] validated the protocol, and the findings were extended to a live confocal microscopy method. • It requires a Zeiss LSM 900 confocal microscope and appropriate imaging software and is optimized for the latest version of Zen Blue. • Anti-PD-1 antibodies are commonly used in cancer therapies, and this protocol optimizes the analysis of their effectiveness.