Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.
CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.
CD19 CAR-T细胞在B淋巴细胞白血病的临床中展现了效力。虽然大多数的携带CD28或者4-1BB的二代CARs已经在病人中进行了研究,但是临床前研究显示同时携带CD28和4-1BB三代(3G)CARs具有更强的能力。然而，对于CARs下游的细胞内信号途径的了解所致甚少。在之前的工作中，我们分析了2G和3G CARs刺激后的信号。3G CARs对于IL-2和自体B细胞的反复刺激，扩增能力比2G CAR T细胞更强。反复的刺激之后，2G和3G CAR-T 细胞都具有持续的细胞毒性。细胞内信号蛋白抗原刺激后的磷酸化状态显示，
被激活，例如参与细胞周期，细胞粘附和胞吐作用的蛋白。总之，3G CAR-T细胞比2G CAR-T细胞具有更高的细胞内信号活性，