{"doi":"10.21769/bioprotoc.5057","title":"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","abstract":"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.","journal":"BIO-PROTOCOL","year":2024,"id":501943,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9596,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":715551,"name":"Shalom Lerrer","orcid":"0000-0003-3416-8568","position":1,"is_corresponding":false},{"id":382487,"name":"Adam Mor","orcid":"0000-0002-2854-2405","position":2,"is_corresponding":false},{"id":1351771,"name":"Justin C Zhong","orcid":null,"position":0,"is_corresponding":true}],"reference_count":21,"raw_metadata":null,"created_at":"2026-07-19T02:10:19.647285Z","pmid":"39282229","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}