Abstract：
Immune cell-based therapies can induce potent antitumor effects but are also often associated with severe toxicities. We previously developed a PD-1-based small molecule-regulated reversible T cell activation switch to control the activity of cellular immunotherapy products. This chemically regulated and SH2-delivered-inhibitory tail (CRASH-IT) switch relies on the noncovalent interaction of switch SH2 domains with phosphorylated ITAM motifs in either chimeric antigen receptors or T cell receptors. After this interaction, the immunoreceptor tyrosine-based inhibition motif/switch motif (ITIM/ITSM) containing PD-1 domain present in the CRASH-IT switch induces robust inhibition of T cell signaling, and CRASH-IT-mediated suppression of T cell activity can be reversed by small molecule-induced switch proteolysis. With the aim to develop improved second-generation switch systems, we here analyze the possibility space of both the immune cell receptor docking and inhibitory signaling domains that allow control over T cell activity. Importantly, these analyses demonstrate that the inhibitory domains that most potently suppress antigen receptor signaling in primary human T cells are not derived from inhibitory receptors, such as PD-1 and BTLA, that are endogenously expressed in T cells, but include ITIM/ITSM containing inhibitory domains derived from receptors present in myeloid cells. In addition, we demonstrate that physical proximity of the inhibitory domain to the antigen receptor is crucial to efficiently suppress T cell activation, as only switch designs that employ SH2 domains directly interacting with ITAM motifs in antigen receptors efficiently and reversibly inhibit T cell functionality. These data demonstrate the flexible and interchangeable nature of immune cell signaling domains, and inform the design of a synthetic proximity-based switch system with a superior dynamic range.
摘要：
基于免疫细胞的疗法可以诱导有效的抗肿瘤作用，但也经常与严重的毒性有关。我们之前开发了一种基于PD-1的小分子调节的可逆性T细胞激活开关，以控制细胞免疫治疗产品的活性。这种化学调节的和SH2递送的抑制性尾部（CRASH-IT）开关依赖于开关SH2结构域与嵌合抗原受体或T细胞受体中磷酸化ITAM基序的非共价相互作用。经过这种互动,基于免疫受体酪氨酸的抑制基序/开关基序（ITIM/ITSM）包含存在于CRASH-IT开关中的PD-1结构域诱导T细胞信号传导的强烈抑制，和CRASH-IT介导的T细胞活性抑制可以通过小分子诱导的开关蛋白水解来逆转。为了开发改进的第二代开关系统，我们在这里分析了允许控制T细胞活性的免疫细胞受体对接和抑制性信号传导结构域的可能性空间.重要的是,这些分析表明，在原代人T细胞中最有效抑制抗原受体信号传导的抑制性结构域不是来自抑制性受体，如PD-1和BTLA，在T细胞中内源性表达，但包括含有来自骨髓细胞中存在的受体的抑制性结构域的ITIM/ITSM。此外,我们证明了抑制性结构域与抗原受体的物理接近对于有效抑制T细胞活化至关重要，仅使用SH2结构域直接与抗原受体中的ITAM基序相互作用的开关设计有效且可逆地抑制T细胞功能。这些数据证明了免疫细胞信号结构域的灵活和可互换的性质，并告知具有优越动态范围的合成接近开关系统的设计。
