Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2022;2446:451-468. doi: 10.1007/978-1-0716-2075-5_23.
Chimeric antigen receptors (CARs) are engineered fusion proteins constructed from antigen-recognition, signaling, and costimulatory domains. CARs can be expressed in T cells with the purpose of reprogramming the T cells to specifically target tumor cells. This strategy thereby avoids the requirement for antigen processing and presentation by the target cell. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan with highly tumor-specific expression in neuroblastoma compared with nonmalignant cells. Therefore, GPC2 is an attractive target candidate for CAR T-cell therapy. Single-domain antibodies (sdAbs) can access epitopes different from those targeted by single-chain variable fragments and, because of their stability and modularity, could serve as ideal antigen-recognition domains in CAR T cells. Here, we describe a protocol for generating GPC2-targeted sdAb CAR T cells. We also present a methodology for assessing the efficiency of CAR expression on human T cells and their ability to kill GPC2-positive neuroblastoma cells in vitro and in vivo. The method described here is applicable to the production of CAR T cells derived from all types of sdAbs including VHHs and VNARs.
嵌合抗原受体（CARs）是由抗原识别、信号和共刺激结构域组成的工程融合蛋白。CARs 可在 T 细胞中表达，目的是对 T 细胞进行重新编程，使其能够特异性靶向肿瘤细胞。这样就避免了靶细胞对抗原的加工和呈递的要求。Glypican-2（GPC2）是一种细胞表面硫酸乙酰肝素蛋白聚糖，与非恶性细胞相比，神经母细胞瘤中高度特异性表达。因此，GPC2 是 CAR T 细胞治疗的一个有吸引力的靶候选物。单域抗体（sdAb）可以访问不同于单链可变片段靶向的表位，并且由于其稳定性和模块化，可作为 CAR T 细胞中理想的抗原识别结构域。在这里，我们描述了一种生成靶向 GPC2 的 sdAb CAR T 细胞的方案。我们还介绍了一种评估 CAR 在人 T 细胞上的表达效率及其在体外和体内杀伤 GPC2 阳性神经母细胞瘤细胞的能力的方法。这里描述的方法适用于所有类型的 sdAb（包括 VHH 和 VNAR）衍生的 CAR T 细胞的生产。
