Protocol to generate human stem cell-derived CD70-directed allogeneic CAR-NKT cells for treating renal cell carcinoma

CD70 has emerged as a promising immunotherapeutic target in renal cell carcinoma (RCC), with both CD70-directed monoclonal antibodies and chimeric antigen receptor (CAR)-based therapies currently under development. Here, we describe a protocol for the generation of human CD70-directed allogeneic CAR-natural killer T (NKT) ( Allo CAR70-NKT) cells derived from cord blood CD34 + hematopoietic stem and progenitor cells (HSPCs) using a clinically guided culture. Furthermore, we describe the therapeutic efficacy of Allo CAR70-NKT cells in mediating cytotoxic activity against RCC cell lines in vitro . For complete details on the use and execution of this protocol, please refer to Li et al. 1 • Generation of Allo CAR70-NKT cells from hematopoietic stem and progenitor cells • Ex vivo differentiation of Allo CAR70-NKT cells via a clinically guided culture method • Evaluation of antitumor capacity of Allo CAR70-NKT cells against renal cell carcinoma Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. CD70 has emerged as a promising immunotherapeutic target in renal cell carcinoma (RCC), with both CD70-directed monoclonal antibodies and chimeric antigen receptor (CAR)-based therapies currently under development. Here, we describe a protocol for the generation of human CD70-directed allogeneic CAR-natural killer T (NKT) ( Allo CAR70-NKT) cells derived from cord blood CD34 + hematopoietic stem and progenitor cells (HSPCs) using a clinically guided culture. Furthermore, we describe the evaluation of the therapeutic efficacy of Allo CAR70-NKT cells in mediating cytotoxic activity against RCC cell lines in vitro .