利用基因工程改造的 HSP 细胞与无饲养层分化培养生成用于现货型肿瘤免疫治疗的同种异体 CAR-NKT 细胞

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 ( Allo NKT) cells and their CAR-armed derivatives ( Allo CAR-NKT cells).

We include detailed information on lentivirus generation and titration, as well as the five stages of ex vivo culture required to generate Allo CAR-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 Allo CAR-NKT cells. It takes ~2 weeks to generate and titrate lentiviruses and ~6 weeks to generate mature Allo CAR-NKT cells. Competence with human stem cell and T cell culture, gene engineering and flow cytometry is required for optimal results.