Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture

The clinical potential of current chimeric antigen receptor-engineered T (CAR-T) cell therapy is hampered by its autologous nature that poses considerable challenges in manufacturing, costs and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture method to differentiate gene-engineered hematopoietic stem and progenitor (HSP) cells into allogeneic invariant natural killer T (^AlloNKT) cells and their CAR-armed derivatives (^AlloCAR-NKT cells). We include detailed information on lentivirus generation and titration, as well as the five stages of ex vivo culture required to generate ^AlloCAR-NKT cells, including HSP cell engineering, HSP cell expansion, NKT cell differentiation, NKT cell deep differentiation and NKT cell expansion. In addition, we describe procedures for evaluating the pharmacology, antitumor efficacy and mechanism of action of ^AlloCAR-NKT cells. It takes ~2 weeks to generate and titrate lentiviruses and ~6 weeks to generate mature ^AlloCAR-NKT cells. Competence with human stem cell and T cell culture, gene engineering and flow cytometry is required for optimal results.