Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling", a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B-cell activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cells signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. Additionally, we observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B-cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that "CAR Pooling" is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

嵌合抗原受体（chimeric antigen receptor, CAR）通过重编程天然信号通路组分，将T细胞重定向至难治性肿瘤，但在持续性、复发性恶性肿瘤中疗效有限。本研究提出“CAR集合筛选（CAR Pooling）”这一多重筛选策略，可快速筛选出具有临床转化潜力的CAR设计。本研究构建了40种携带源自不同免疫细胞谱系信号结构域的CAR，通过混合筛选实验评估它们在模拟长期肿瘤抗原暴露的反复刺激过程中，激活关键T细胞效应功能的能力。研究从主要与B细胞相关的肿瘤坏死因子（tumor necrosis factor, TNF）受体家族中，筛选得到多个有效信号结构域：CD40可增强T细胞增殖，而B细胞活化因子受体（B-cell activating factor receptor, BAFF-R）与跨膜激活剂及CAML相互作用因子（transmembrane activator and CAML interactor, TACI）则可促进细胞毒性效应。相较于临床常用基准CAR，这些结构域介导的信号通路在长期抗原刺激后可增强上述功能，且通过这些结构域传导信号的CAR-T细胞可呈现出记忆、细胞毒性及代谢特征各异的细胞状态。表达BAFF-R的CAR-T细胞富集有此前被证实与临床良好预后相关的高细胞毒性转录特征。此外，本研究发现，在经临床验证的B细胞成熟抗原（B-cell maturation antigen, BCMA）靶向CAR中，用BAFF-R胞内信号结构域替换4-1BB胞内信号结构域，可在多发性骨髓瘤异种移植模型中增强CAR-T细胞的抗肿瘤活性。综上，本研究结果表明，“CAR集合筛选（CAR Pooling）”是一种可快速探索CAR结构与功能、进而提升CAR-T细胞治疗疗效的通用策略。