Engineering potent chimeric antigen receptor T cells by programming signaling during T-cell activation [RNA-seq]. Engineering potent chimeric antigen receptor T cells by programming signaling during T-cell activation [RNA-seq]

Programming cell signaling during T-cell activation represents a simple strategy for improving the potency of therapeutic T-cell products. Stim-R™ technology (Lyell Immunopharma) is a customizable, degradable synthetic cell biomimetic that emulates physiologic, cell-like presentation of signal molecules to control T-cell activation. A breadth of Stim-R formulations with different anti-CD3/anti-CD28 (αCD3/αCD28) antibody densities and stoichiometries were screened for their effects on multiple metrics of T-cell function. We identified an optimized formulation that produced receptor tyrosine kinase-like orphan receptor 1 (ROR1)-targeted chimeric antigen receptor (CAR) T cells with enhanced persistence and polyfunctionality in vitro, as assessed in repeat-stimulation assays, compared with a benchmark product generated using a conventional T-cell–activating reagent. In transcriptomic analyses, CAR T cells activated with Stim-R technology showed downregulation of exhaustion-associated gene sets and retained a unique subset of stem-like cells with effector-associated gene signatures following repeated exposure to tumor cells. Compared with the benchmark product, CAR T cells activated using the optimized Stim-R technology formulation exhibited higher peak expansion, prolonged persistence, and improved tumor control in a solid tumor xenograft model. Enhancing T-cell products with Stim-R technology during T-cell activation may help improve therapeutic efficacy against solid tumors. Overall design: Human CAR T cells produced using the optimized Stim-R formulation or the benchmark reagent were collected at the end of the third stimulation with H1975 tumor cells (Day 10) in the normalized repeat-stimulation assay. Live CD45+EGFR+CD8+ T cells were isolated by flow sorting for bulk RNA-Seq profiling.

在T细胞活化过程中编程细胞信号通路，是提升治疗性T细胞产品效能的一种简便策略。Stim-R™技术（Lyell Immunopharma）是一种可定制、可降解的合成细胞仿生体系，可模拟生理状态下的细胞样信号分子呈递，以此调控T细胞活化。我们针对一系列具有不同抗CD3/抗CD28（αCD3/αCD28）抗体密度与化学计量比的Stim-R制剂，筛选其对T细胞功能多项指标的影响。相较于采用常规T细胞活化试剂制备的基准产品，我们筛选得到了一款优化制剂，其所制备的靶向受体酪氨酸激酶样孤儿受体1（ROR1）的嵌合抗原受体（CAR）T细胞，在体外重复刺激实验中展现出更强的存续能力与多功能性。转录组分析结果显示，经Stim-R技术活化的CAR-T细胞在反复接触肿瘤细胞后，耗竭相关基因集表达下调，并保留了具有效应功能基因特征的独特干细胞样细胞亚群。与基准产品相比，采用优化后Stim-R技术制剂活化的CAR-T细胞，在实体瘤异种移植模型中展现出更高的峰值扩增效率、更持久的存续能力以及更优异的肿瘤控制效果。在T细胞活化阶段通过Stim-R技术优化T细胞产品，或可提升其针对实体瘤的治疗效能。实验整体设计：在标准化重复刺激实验中，将采用优化后Stim-R制剂或基准试剂制备的人源CAR-T细胞，在第三次与H1975肿瘤细胞共刺激结束后（第10天）收集样本。通过流式分选获取活的CD45+EGFR+CD8+ T细胞，用于批量RNA测序（bulk RNA-Seq）分析。