The immunostimulatory intracellular domains (ICDs) of chimaeric antigen receptors (CARs) are essential for converting antigen recognition into antitumoural function. Although there are many possible combinations of ICDs, almost all current CARs rely on combinations of CD3𝛇, CD28 and 4-1BB. Here we show that a barcoded library of 700,000 unique CD19-specific CARs with diverse ICDs cloned into lentiviral vectors and transduced into Jurkat T cells can be screened at high throughput via cell sorting and next-generation sequencing to optimize CAR signalling for antitumoural functions. By using this screening approach, we identified CARs with new ICD combinations that, compared with clinically available CARs, endowed human primary T cells with comparable tumour control in mice and with improved proliferation, persistence, exhaustion and cytotoxicity after tumour rechallenge in vitro. The screening strategy can be adapted to other disease models, cell types and selection conditions, and could be used to improve adoptive cell therapies and to expand their utility to new disease indications.

嵌合抗原受体 (CAR) 的免疫刺激细胞内结构域 (ICD) 对于将抗原识别转化为抗肿瘤功能至关重要。虽然 ICD 有很多可能的组合，但目前几乎所有的 CAR 都依赖于 CD3 𝛇的组合、CD28 和 4-1BB。在这里，我们展示了一个包含 700,000 个独特的 CD19 特异性 CAR 的条形码库，这些 CAR 具有不同的 ICD，克隆到慢病毒载体中并转导到 Jurkat T 细胞中，可以通过细胞分选和下一代测序以高通量筛选，以优化 CAR 信号传导以实现抗肿瘤功能。通过使用这种筛选方法，我们确定了具有新 ICD 组合的 CAR，与临床可用的 CAR 相比，这些组合赋予人类原代 T 细胞在小鼠中具有可比的肿瘤控制能力，并且在体外肿瘤再攻击后具有改善的增殖、持久性、衰竭和细胞毒性。筛选策略可以适应其他疾病模型、细胞类型和选择条件，并可用于改进过继细胞疗法并将其效用扩展到新的疾病适应症。