Abstract LB123: A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways

A. Maurin, C. Franchet, S. Schann, X. Leroy
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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.
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LB123:一个高价值的药理学平台,致力于刺激免疫检查点信号通路的实时研究
免疫检查点(ICP)共受体在免疫应答的精细调节中起着关键作用。抑制性ICP共受体在癌症中主要表现为免疫反应的衰竭,而刺激性ICP共受体显然参与了导致自身免疫性疾病的免疫系统的无序激活。但另一方面,这种共受体亚型通过增加抗肿瘤反应在癌症治疗中显示出巨大的价值。总之,这些共受体对免疫学或免疫肿瘤学的创新疗法的发展表现出极大的兴趣。目前,针对这些共受体的治疗方法正在临床试验中进行测试或被批准用于人体,特别是针对抑制性和刺激性ICP的抗体。基于小分子的策略在早期药物发现和早期临床试验中得到检验。为了筛选和评估这些疗法的效力和效率,迫切需要强大而可靠的基于细胞的检测。在Domain Therapeutics,使用我们强大的专有BRET技术:bioSens-AllTM,我们成功开发并验证了基于细胞的检测平台,以评估免疫检查点药理学。在活细胞中实时监测检查点共受体信号的激活或阻断。设计了专门用于刺激icp的BRET试验:4-1BB/4-1BB配体和OX-40/OX-40配体。BRET检测发展的灵感来自于在激活途径中非常接近其共受体的特定细胞质伴侣的自然招募和邻近性。事实上,第一个检测设计是基于与rGFP融合的共受体和与rLucII融合的细胞质伴侣。在第二种实验设计中,共受体不再融合,而是与rGFP融合的膜锚共转染,与rLucII融合的细胞质伴侣共转染。因此,在活化系统(可溶性配体、抗体或共培养系统)中,可以观察到BRET的调节。对于专用于4-1BB的ICP分析,使用可溶性配体、激动剂抗体或TRAF1与rLucII融合的生物传感器共培养来触发BRET信号。进一步验证了OX-40/OX-40配体。这些BRET检测补充了先前开发的抑制性icp轴:PD-1/ pd -配体1或pd -配体2和CTLA-4/CD80-CD86。本文建立的ICP平台具有良好的准确性和鲁棒性,是一种强大可靠的ICP药物发现技术。我们基于细胞的时空功能分析可以支持广泛的药物项目,包括:高通量功能筛选、先导物优化和生物分析QC批放行。引文格式:Alice GENTIL DIT MAURIN, Christel FRANCHET, Stephan SCHANN, Xavier LEROY。一个高价值药理学平台,致力于刺激免疫检查点信号通路的实时研究[摘要]。见:美国癌症研究协会2021年年会论文集;2021年4月10日至15日和5月17日至21日。费城(PA): AACR;癌症杂志,2021;81(13 -增刊):摘要nr LB123。
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