Protocol for preparing metabolically reprogrammed human CAR T cells and evaluating their in vitro effects

Abstract

Summary: Chimeric antigen receptor (CAR) T cell therapy represents a cutting-edge cancer treatment, making the development and testing of CAR T cells crucial for advancing this therapeutic strategy. We present a protocol for creating and characterizing human epidermal growth factor receptor 2 (HER2)- and glypican-3 (GPC3)-metabolic reprogramming (MR)-CAR T cells by overexpressing adenosine deaminase 1 (ADA1) and CD26 (also known as dipeptidylpeptidase-4 or DPP4). This approach effectively converts immunosuppressive adenosine into inosine, which supports T cell survival in glucose-deficient tumor microenvironments. The protocol includes producing retroviral vectors, generating CAR T cells, and conducting ecto-ADA1 activity, cytotoxicity, cell migration, and RNA sequencing assays.For complete details on the use and execution of this protocol, please refer to Hu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• cancer
• clinical protocol
• immunology
• metabolism
• molecular biology
• tissue engineering