A genome-scale gain-of-function CRISPR screen identifies proline metabolism in T cells as a means to enhance CAR-T therapy

Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells, and we identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics revealed that augmenting PRODH2 expression in the T cell re-shaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.

针对癌症与免疫性疾病的嵌合抗原受体（Chimeric antigen receptor, CAR）T细胞免疫疗法已取得长足进展，但仍面临多重阻碍。精准找到可将T细胞工程化以实现预期功能的分子靶点，对于丰富以T细胞为中心的治疗手段库具有广泛意义。本研究在原代CD8+ T细胞中建立了基于失活向导RNA（dead-guide RNA, dgRNA）的CRISPR激活筛选体系，筛选出可用于CAR-T细胞工程化的功能获得性（gain-of-function, GOF）靶点。对候选靶点PRODH2进行靶向敲入后，可在多种癌症模型中增强CAR-T细胞的杀伤活性与体内抗肿瘤效力。转录组学与代谢组学分析显示，在T细胞中上调PRODH2的表达可重塑广泛且独特的基因表达与代谢程序。线粒体、代谢及免疫学分析进一步证实，PRODH2工程化改造可增强CAR-T细胞的抗肿瘤代谢与免疫功能。综上，本研究构建了一套功能获得性免疫增强剂的筛选体系，并证明PRODH2可作为增强CAR-T治疗效力的靶点。