嵌合抗原受体（CAR）-T细胞在实体瘤治疗中面临许多障碍，包括异质抗原表达和低效的T细胞持久性。鸟苷酸环化酶 C (GUCY2C) 已被确定为适合靶向治疗的肿瘤抗原，因为其在正常组织中的肠道限制性表达模式以及在胃肠道肿瘤（尤其是结直肠癌）中的稳定过度表达。本研究研究了一种针对GUCY2C的抗原敏感且长效的CAR-T细胞。利用构建的具有不同GUCY2C表达密度的肿瘤细胞系，我们筛选出一种抗原敏感的单链可变片段（scFv），该单链可变片段（scFv）能够实现CAR-T细胞有效地根除GUCY2C低表达的肿瘤细胞。还构建了具有不同铰链、跨膜和共刺激结构域组成的 CAR-T 细胞，用于选择在体外和荷瘤小鼠中具有持久抗肿瘤功效的持久 CAR-T 形式。基于铰链和跨膜结构域的突变，进一步研究了其潜在机制。我们发现抗原敏感的scFv、CD8α铰链、CD8α跨膜和CD28共刺激结构域的组成增强了CAR-T细胞快速杀伤肿瘤，维持高活性。在各种结直肠癌模型中的扩展能力和长期疗效。持久的抗肿瘤功能归因于最佳的 CAR 补品信号传导，该信号赋予 CAR-T 细胞在没有抗原刺激的情况下自主激活、增殖、存活和细胞因子释放的能力。强直信号与 CD8α 铰链和跨膜结构域中的长度和半胱氨酸残基相关。这项研究证明了一种有效的 GUCY2C 靶向 CAR-T 细胞用于胃肠道肿瘤治疗，并强调了足够的强直信号对于有效 CAR-T 细胞的重要性实体瘤治疗。© 作者（或其雇主）2024。根据 CC BY-NC 允许重复使用。禁止商业再利用。请参阅权利和权限。英国医学杂志出版。

Chimeric antigen receptor (CAR)-T cells face many obstacles in solid tumor therapy, including heterogeneous antigen expression and inefficient T cell persistence. Guanylyl cyclase C (GUCY2C) has been identified as a suitable tumor antigen for targeted therapy due to its intestinal-restricted expression pattern in normal tissues and steady overexpression in gastrointestinal tumors, especially colorectal cancer. An antigen-sensitive and long-lasting CAR-T cell targeting GUCY2C was investigated in this study.Using constructed tumor cell lines with various GUCY2C expression densities, we screened out an antigen-sensitive single chain variable fragment (scFv) that enabled CAR-T cells to efficiently eradicate the GUCY2C lowly expressed tumor cells. CAR-T cells with different compositions of the hinge, transmembrane and costimulatory domains were also constructed for selection of the long-lasting CAR-T format with durable antitumor efficacy in vitro and in tumor-bearing mice. The underlying mechanism was further investigated based on mutation of the hinge and transmembrane domains.We found that the composition of the antigen-sensitive scFv, CD8α hinge, CD8α transmembrane, and CD28 costimulatory domains boosted CAR-T cells to rapidly kill tumors, maintain high expansion capacity, and long-term efficacy in various colorectal cancer models. The durable antitumor function was attributed to the optimal CAR tonic signaling that conferred CAR-T cells with autonomous activation, proliferation, survival and cytokine release in the absence of antigen stimulation. The tonic signaling was associated with the length and the cysteine residues in the CD8α hinge and transmembrane domains.This study demonstrated a potent GUCY2C-targeted CAR-T cell for gastrointestinal tumor therapy and highlights the importance of adequate tonic signaling for effective CAR-T cell therapy against solid tumors.© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.