肿瘤细胞治疗研究
英文原题：CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells.
CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells.
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我们得出结论，CAR 的分子结构、膜组织和流动性是关键的设计参数，能够预测有效免疫突触的形成。
嵌合抗原受体（CAR）修饰的T细胞为化疗耐药淋巴瘤的治疗带来了范式转变。相反，针对实体瘤的CAR-T 细胞临床经验令人沮丧，表明有必要对其进行分子层面的优化。虽然在CD3z信号结构域之外将CD28或41BB共刺激结构域纳入CAR中改善了T细胞产物的长期疗效，但它们对早期肿瘤接合的影响尚未阐明。我们使用超分辨率AiryScan显微镜和荧光相关光谱研究了静息T细胞膜中第一代（.z）、第二代（CD28.z、41BB.z）和第三代（CD28.41BB.z）HER2特异性CAR的抗原非依赖性自缔合和膜扩散动力学，并与基于RoseTTAFold的结构预测和天然Western blot中寡聚化的评估相关联。.z和CD28.z CAR形成了大的、高密度的亚微米二聚体簇，而含41BB的CAR形成更高的寡聚体，组装成更小但数量更多的膜簇。第一代、第二代和第三代CAR随着其CD3z结构域距膜平面的距离增加，表现出逐渐增加的侧向扩散。免疫突触的共聚焦显微镜分析显示，高度移动的CD28.41BB.z的小簇和移动性较低的.z CAR的大簇均比中等移动性的CD28.z或41BB.z CAR诱导更有效的CD3和pLck磷酸化。然而，电细胞-基底阻抗传感揭示，CD28.41BB.z CAR在连续短期消除贴壁肿瘤细胞方面表现最差，而.z CAR优于所有其他CAR。我们得出结论，CAR的分子结构、膜组织和流动性是关键的设计参数，能够预测有效免疫突触的形成。因此，在追求最佳治疗效果时，需要将这些因素与共刺激域的长期生物学效应一并考虑。
Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3 and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.
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