Henghui Li, Jingyao Li, Ying Wang, Liang Chen
Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable clinical success in hematological malignancies; however, its broader application is limited by high manufacturing costs and insufficient efficacy against solid tumors. To overcome these challenges, an in situ CAR-T cell generation strategy was developed based on lipid nanoparticle (LNP)-mediated delivery of CAR mRNA targeting Claudin18.2, enabling the transient production of CAR-T cells directly within tumor tissue. Following LNP transfection, activated T cells exhibited efficient surface expression of CARs. These transient CAR-T cells demonstrated potent cytotoxic activity against Claudin18.2-positive gastric carcinoma cell lines, including NUGC4 and NCI-N87, and produced significant antitumor efficacy in tumor-bearing mouse models. This protocol describes detailed procedures for in vitro transcription of CAR mRNA, LNP formulation, T-cell activation and transfection, CAR expression analysis, and in vitro and in vivo functional evaluation of CAR-T-mediated cytotoxicity. Taken together, this strategy provides a cost-effective and versatile platform for generating transient CAR-T cells and offers a promising approach for treating solid tumors.