Epigenetically programmed 'stem-like' tissue resident memory CAR T-cells exhibit robust activity against solid and liquid tumors [scRNA]

Chimeric antigen receptor (CAR) T-cells have shown remarkable success in the therapy of hematological malignancies, but they have not yet proven nearly as effective in treating non-hematopoietic cancers. This study proposes augmentation of CAR T-cell function and accumulation in solid tumors by modifying the epigenetic landscape that governs early memory differentiation and adaptation to tissue residency. We identified that a key factor in human tissue-resident memory CAR T-cell (CAR-TRM) formation is activation in the presence of the pleotropic cytokine, TGF-ß, which epigenetically enforces a core transcriptional program of both 'stemness' and sustained tissue residency. This strategy results in a readily actionable in vitro production method for engineering peripheral blood T-cells into large numbers of 'stem-like' CAR-TRM cells resistant to epigenetically-imposed exhaustion that possess enhanced ability to accumulate in situ and rapidly kill cancer cells for more effective immunotherapy. Overall design: Anti-mesothelin M5 CAR-TCONV and CAR-TRM cells were harvested after nine days of in vitro expansion. Dead cells were eliminated using the Dead Cell Removal Kit (Miltenyi Biotec). Mesothelin-directed M5 CAR-TCONV and -TRM cells were magnetically isolated using anti-mesothelin-coated beads. After isolation, T-cell viability and quantity were evaluated using trypan blue exclusion and cells were resuspended in PBS containing 0.04% BSA.

嵌合抗原受体（Chimeric antigen receptor, CAR）T细胞在血液系统恶性肿瘤的治疗中已展现出卓越疗效，但在非造血系统恶性肿瘤的治疗中尚未展现出相近的有效性。本研究提出通过调控调控早期记忆分化与组织驻留适应性的表观遗传景观，以增强CAR T细胞在实体瘤中的功能与聚集能力。研究发现，人组织驻留记忆CAR T细胞（CAR-TRM）形成的关键因素是在多效性细胞因子转化生长因子β（TGF-β）存在条件下的活化，该因子可通过表观遗传机制强制执行兼具“干性”与持续组织驻留特性的核心转录程序。该策略可开发出一种易于实施的体外生产方法，将外周血T细胞工程改造为大量“干细胞样”CAR-TRM细胞，这类细胞可抵抗表观遗传介导的耗竭，且具备更强的原位聚集能力与快速杀伤癌细胞的能力，从而实现更高效的免疫治疗。
总体实验设计：在体外扩增9天后收获抗间皮素M5常规CAR T细胞（CAR-TCONV）与CAR-TRM细胞。使用死细胞去除试剂盒（Dead Cell Removal Kit, Miltenyi Biotec）清除死细胞。采用抗间皮素包被磁珠磁分选获得靶向间皮素的M5 CAR-TCONV与CAR-TRM细胞。分选完成后，通过台盼蓝拒染法评估T细胞活力与数量，并将细胞重悬于含0.04%牛血清白蛋白（Bovine Serum Albumin, BSA）的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中。