{"title":"Abstract LB123: A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways","authors":"A. Maurin, C. Franchet, S. Schann, X. Leroy","doi":"10.1158/1538-7445.AM2021-LB123","DOIUrl":null,"url":null,"abstract":"Immune checkpoint (ICP) co-receptors play a key role in the fine modulation of the immune response. While inhibitory ICP co-receptors are mainly characterized in cancer by the exhaustion of the immune response, stimulatory ICP co-receptors are clearly involved in an anarchic activation of the immune system leading to autoimmune diseases. But in another hand, this subtype of co-receptor shows a great asset in cancer therapy by increasing anti-tumoral response. All together, these co-receptors show a great interest for the development of innovative therapies in immunology or immuno-oncology. Nowadays, treatments targeting these co-receptors are currently tested in clinical trials or approved for use in man, especially antibodies targeting both inhibitory and stimulatory ICP. Small molecule-based strategy is tested in early drug discovery and in early clinical trials. In order to screen and assess the potency and efficiency of these therapies, robust and reliable cell-based assays are urgently needed. At Domain Therapeutics, using our powerful proprietary BRET technology: bioSens-AllTM, we successfully develop and validate a cell-based assay platform to assess immune checkpoint pharmacology. The activation or blockade of the signaling of checkpoint co-receptors are monitored in real time and in living cells. BRET assays dedicated to stimulatory ICPs: 4-1BB/4-1BB Ligand and OX-40/OX-40 Ligand were designed. BRET assay development is inspired by the natural recruitment and proximity of specific cytoplasmic partners very close to their co-receptors in an activated pathway. Indeed, the first assay design is based on the co-receptor fused with rGFP and its cytoplasmic partner fused with a rLucII. In the second assay design, the co-receptor is no longer fused, but is co-transfected with a membrane anchor fused with the rGFP and co-transfected with the cytoplasmic partner fused with rLucII. So, in an activated system (soluble ligand, antibody or in co-culture system) a modulation of BRET can be observed. For ICP assays dedicated to 4-1BB, BRET signal is triggered using soluble ligand, agonist antibody, or co-culture where TRAF1 is the biosensor fused with rLucII. Further validation was obtained for OX-40/OX-40 Ligand. Those BRET assays complement once previously developed for inhibitory ICPs axis: PD-1/PD-Ligand 1 or PD-Ligand 2 and CTLA-4/CD80-CD86. The ICP Platform set up here shows a good accuracy and robustness and represents a strong and reliable technology for drug discovery dedicated to ICPs. Our spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Citation Format: Alice GENTIL DIT MAURIN, Christel FRANCHET, Stephan SCHANN, Xavier LEROY. A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB123.","PeriodicalId":12258,"journal":{"name":"Experimental and Molecular Therapeutics","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Experimental and Molecular Therapeutics","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1158/1538-7445.AM2021-LB123","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Immune checkpoint (ICP) co-receptors play a key role in the fine modulation of the immune response. While inhibitory ICP co-receptors are mainly characterized in cancer by the exhaustion of the immune response, stimulatory ICP co-receptors are clearly involved in an anarchic activation of the immune system leading to autoimmune diseases. But in another hand, this subtype of co-receptor shows a great asset in cancer therapy by increasing anti-tumoral response. All together, these co-receptors show a great interest for the development of innovative therapies in immunology or immuno-oncology. Nowadays, treatments targeting these co-receptors are currently tested in clinical trials or approved for use in man, especially antibodies targeting both inhibitory and stimulatory ICP. Small molecule-based strategy is tested in early drug discovery and in early clinical trials. In order to screen and assess the potency and efficiency of these therapies, robust and reliable cell-based assays are urgently needed. At Domain Therapeutics, using our powerful proprietary BRET technology: bioSens-AllTM, we successfully develop and validate a cell-based assay platform to assess immune checkpoint pharmacology. The activation or blockade of the signaling of checkpoint co-receptors are monitored in real time and in living cells. BRET assays dedicated to stimulatory ICPs: 4-1BB/4-1BB Ligand and OX-40/OX-40 Ligand were designed. BRET assay development is inspired by the natural recruitment and proximity of specific cytoplasmic partners very close to their co-receptors in an activated pathway. Indeed, the first assay design is based on the co-receptor fused with rGFP and its cytoplasmic partner fused with a rLucII. In the second assay design, the co-receptor is no longer fused, but is co-transfected with a membrane anchor fused with the rGFP and co-transfected with the cytoplasmic partner fused with rLucII. So, in an activated system (soluble ligand, antibody or in co-culture system) a modulation of BRET can be observed. For ICP assays dedicated to 4-1BB, BRET signal is triggered using soluble ligand, agonist antibody, or co-culture where TRAF1 is the biosensor fused with rLucII. Further validation was obtained for OX-40/OX-40 Ligand. Those BRET assays complement once previously developed for inhibitory ICPs axis: PD-1/PD-Ligand 1 or PD-Ligand 2 and CTLA-4/CD80-CD86. The ICP Platform set up here shows a good accuracy and robustness and represents a strong and reliable technology for drug discovery dedicated to ICPs. Our spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Citation Format: Alice GENTIL DIT MAURIN, Christel FRANCHET, Stephan SCHANN, Xavier LEROY. A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB123.