Department of Chemical and Biomolecular Engineering, University of California, Los Angeles (UCLA), Los Angeles, CA, USA.
Department of Medicine, Washington University in St. Louis, St. Louis, MO, USA.
Nat Protoc. 2020 Apr;15(4):1507-1524. doi: 10.1038/s41596-020-0294-8. Epub 2020 Feb 26.
The expression of synthetic receptors in primary T cells enables the programming of user-defined responses when designing T-cell therapies. Chimeric antigen receptors (CARs) are synthetic receptors that have demonstrated efficacy in cancer therapy by targeting immobilized antigens on the surface of malignant cells. Recently, we showed they can also rewire T-cell responses to soluble ligands. In contrast to other synthetic receptors, CARs are not only readily engineered by rational design, but also clinically translatable, with robust function in primary human T cells. This protocol discusses design principles for CARs responsive to soluble ligands and delineates steps for producing T cells expressing synthetic receptors. Functional assays for quantifying the ability of CAR T cells to sense and respond to soluble ligands are also presented. This protocol provides a framework for proficient immune-cell researchers to test novel T-cell therapies targeting soluble ligands in <2 weeks.
在设计 T 细胞疗法时，将合成受体表达于原代 T 细胞中可以实现用户定义的反应编程。嵌合抗原受体 (CAR) 是一种合成受体，通过靶向恶性细胞表面固定的抗原，在癌症治疗中已显示出疗效。最近，我们发现它们还可以重新连接 T 细胞对可溶性配体的反应。与其他合成受体不同，CAR 不仅可以通过合理设计进行轻松设计，而且具有临床转化潜力，在原代人 T 细胞中具有强大的功能。本方案讨论了响应可溶性配体的 CAR 的设计原则，并描述了表达合成受体的 T 细胞的制备步骤。还介绍了用于定量测定 CAR T 细胞感知和响应可溶性配体能力的功能测定方法。本方案为熟练的免疫细胞研究人员提供了一个在<2 周内测试针对可溶性配体的新型 T 细胞疗法的框架。
