Development of Glypican-2 Targeting Single-Domain Antibody CAR T Cells for Neuroblastoma

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.