CAR-T细胞疗法的一个关键是CAR的设计。目前FDA批准的CAR-T产品仍具有复发等问题。申请人之前发现CAR的活化潜能能影响T细胞分化并找到了疗效更好的新CAR。但CAR的设计如何影响活化潜能及疗效还缺乏研究。本项目拟用基于荧光共振能量转移（FRET）的生物传感器作为工具来测量原代人T细胞中CAR的活化潜能，通过活细胞成像监测T细胞活化通路中Ca2+、ZAP-70或Erk等关键信号的活性。先用二代CD19CAR的模型确定这些生物传感器与活化潜能以及疗效的关联。再用它们来测量新设计的三代CD19CAR以及二代CD22CAR的活化潜能并通过动物模型验证其疗效，找到最优CAR的设计应用于临床，并阐明设计原理。本研究将建立筛选CAR的FRET生物传感器工具，改变当前基于试错的CAR的设计方法。用基于设计原理的方法来工程化改造CAR可以提高成功率，缩短临床前研究从而加快CAR-T细胞疗法的转化。

A key to CAR-T cell therapy’s success is the design of CAR. Current two FDA-approved CAR-T products still have efficacy and relapse problems, with room for further engineering and improvement. Our previous work on a 1928 second generation CAR showed that the activation potential of CAR directs T cell fate and discovered a new CAR (1XX) with enhanced therapeutic potency. We hypothesized that the activation potential (AP) of a CAR design could generally be correlated with its therapeutic potency (TP). However, there is no established method to quantify CAR’s AP. We propose to use fluorescence resonance energy transfer (FRET)-based biosensors as quantitative tools to measure AP of CARs in primary human T cells. Three FRET biosensors that can monitor Ca2+, ZAP-70 or Erk activity in single cells, which are critical signals in T cell activation pathways, will be utilized through live cell imaging technologies. First, we will use CARs from our previous work (1928,1XX and XX3) to establish which FRET-based biosensors can be used as FRET-meters to measure AP and establish standard curves that correlate AP and TP. Then we will use these FRET-meters to measure AP of other CD19-targeting second generation CARs with different ScFv and hinge domains and use the above standard curves to predict their TP, which will be validated through in vivo mouse model. Then we will use FRET biosensors to optimize third generation CD19 CARs as well as CD22 CARs, validating the general applicability of these design principles. Through multiple rounds of FRET-based rational design and validation, we will not only establish FRET biosensor tools for CAR application, we can also change current “try and error”-based methods to design-principle based engineering to optimize other CARs targeting both hematopoietic malignancies and solid tumors. Thus, our study would help shorten the preclinical research and speed up the clinical translation of CAR-T cell therapy.