Chimeric antigen receptor (CAR)-T cells have demonstrated significant clinical potential; however, their strong antitumor activity may cause severe adverse effects. To ensure efficacy and safe CAR-T cell therapy, it is important to understand CAR's structure-activity relationship. To clarify the role of hinge and transmembrane domains in CAR and CAR-T cell function, we generated different chimeras and analyzed their expression levels and antigen-specific activity on CAR-T cells. First, we created a basic CAR with hinge, transmembrane, and signal transduction domains derived from CD3ζ, then we generated six CAR variants whose hinge or hinge/transmembrane domains originated from CD4, CD8α, and CD28. CAR expression level and stability on the T cell were greatly affected by transmembrane rather than hinge domain. Antigen-specific functions of most CAR-T cells depended on their CAR expression levels. However, CARs with a CD8α- or CD28-derived hinge domain showed significant differences in CAR-T cell function, despite their equal expression levels. These results suggest that CAR signaling intensity into T cells was affected not only by CAR expression level, but also by the hinge domain. Our discoveries indicate that the hinge domain regulates the CAR signaling threshold and the transmembrane domain regulates the amount of CAR signaling via control of CAR expression level.

中文翻译：

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嵌合抗原受体的铰链和跨膜结构域调节受体表达和信号阈值。

嵌合抗原受体（CAR）-T细胞已显示出显着的临床潜力。但是，它们强大的抗肿瘤活性可能会导致严重的不良反应。为了确保疗效和安全的CAR-T细胞疗法，了解CAR的结构-活性关系非常重要。为了阐明铰链和跨膜结构域在CAR和CAR-T细胞功能中的作用，我们产生了不同的嵌合体，并分析了它们在CAR-T细胞上的表达水平和抗原特异性活性。首先，我们创建了一个具有衍生自CD3ζ的铰链，跨膜和信号转导域的基本CAR，然后我们生成了六个CAR变体，它们的铰链或铰链/跨膜域均源自CD4，CD8α和CD28。跨膜而非铰链结构域极大地影响了T细胞上CAR表达水平和稳定性。大多数CAR-T细胞的抗原特异性功能取决于其CAR表达水平。但是，具有CD8α或CD28衍生铰链结构域的CAR，尽管它们的表达水平相同，但在CAR-T细胞功能上却表现出显着差异。这些结果表明，CAR进入T细胞的信号强度不仅受CAR表达水平的影响，还受铰链结构域的影响。我们的发现表明铰链结构域通过控制CAR表达水平来调节CAR信号传导阈值，而跨膜结构域调节CAR信号传导的量。这些结果表明，CAR进入T细胞的信号强度不仅受CAR表达水平的影响，还受铰链结构域的影响。我们的发现表明铰链结构域通过控制CAR表达水平来调节CAR信号传导阈值，而跨膜结构域调节CAR信号传导的量。这些结果表明，CAR进入T细胞的信号强度不仅受CAR表达水平的影响，还受铰链结构域的影响。我们的发现表明铰链结构域通过控制CAR表达水平来调节CAR信号传导阈值，而跨膜结构域调节CAR信号传导的量。