{"title":"一种基于成像的检测方法,用于测量 PD-1 在免疫突触中的位置,以测试抗 PD-1 和抗 PD-L1 抗体的结合效力。","authors":"Justin C Zhong, Shalom Lerrer, Adam Mor","doi":"10.21769/BioProtoc.5057","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0000,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11393042/pdf/","citationCount":"0","resultStr":"{\"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.\",\"authors\":\"Justin C Zhong, Shalom Lerrer, Adam Mor\",\"doi\":\"10.21769/BioProtoc.5057\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":93907,\"journal\":{\"name\":\"Bio-protocol\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.0000,\"publicationDate\":\"2024-09-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11393042/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bio-protocol\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.21769/BioProtoc.5057\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21769/BioProtoc.5057","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOLOGY","Score":null,"Total":0}
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.