Chimeric antigen receptors (CARs) consist of an extracellular antigen-binding region fused to intracellular signaling domains, thus enabling customized T cell responses against target cells. Due to the low-throughput process of systematically designing and functionally testing CARs, only a small set of immune signaling domains have been thoroughly explored, despite their major role in T cell activation, effector function and persistence. Here, we present speedingCARs, an integrated method for engineering CAR T cells by signaling domain shuffling and functional screening by single-cell sequencing. Leveraging the inherent modularity of natural signaling domains, we generated a diverse library of 180 unique CAR variants, which were genomically integrated into primary human T cells by CRISPR-Cas9. Functional and pooled screening of the CAR T cell library was performed by co-culture with tumor cells, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), thus enabling high-throughput profiling of multi-dimensional cellular responses. This led to the discovery of several CAR variants that retained the ability to kill tumor cells, while also displaying diverse transcriptional signatures and T cell phenotypes. In summary, speedingCARs substantially expands and characterizes the signaling domain combinations suited for CAR design and supports the engineering of next-generation T cell therapies.

中文翻译：

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speedingCARs：通过信号域改组和单细胞测序加速 CAR T 细胞的工程化

嵌合抗原受体 (CAR) 由与细胞内信号结构域融合的细胞外抗原结合区组成，从而能够针对靶细胞进行定制的 T 细胞反应。由于系统设计和功能测试 CAR 的低通量过程，尽管它们在 T 细胞激活、效应子功能和持久性中起主要作用，但只有一小部分免疫信号域得到了彻底的探索。在这里，我们提出了 speedingCARs，这是一种通过信号域改组和单细胞测序功能筛选来设计 CAR T 细胞的综合方法。利用自然信号域的固有模块性，我们生成了一个包含 180 个独特 CAR 变体的多样化文库，这些变体通过 CRISPR-Cas9 在基因组上整合到原代人类 T 细胞中。通过与肿瘤细胞共培养，然后进行单细胞 RNA 测序 (scRNA-seq) 和单细胞 CAR 测序 (scCAR-seq)，对 CAR T 细胞文库进行功能和合并筛选，从而实现高通量分析多维细胞反应。这导致发现了几种 CAR 变体，它们保留了杀死肿瘤细胞的能力，同时还显示出不同的转录特征和 T 细胞表型。总之，speedingCARs 极大地扩展和表征了适合 CAR 设计的信号域组合，并支持下一代 T 细胞疗法的工程设计。从而实现多维细胞反应的高通量分析。这导致发现了几种 CAR 变体，它们保留了杀死肿瘤细胞的能力，同时还显示出不同的转录特征和 T 细胞表型。总之，speedingCARs 极大地扩展和表征了适合 CAR 设计的信号域组合，并支持下一代 T 细胞疗法的工程设计。从而实现多维细胞反应的高通量分析。这导致发现了几种 CAR 变体，它们保留了杀死肿瘤细胞的能力，同时还显示出不同的转录特征和 T 细胞表型。总之，speedingCARs 极大地扩展和表征了适合 CAR 设计的信号域组合，并支持下一代 T 细胞疗法的工程设计。