Engineering mRNA CAR-T Cells for Cancer Immunotherapy

Adoptive T-cell therapy is a promising cancer immunotherapy, but isolating tumor-specific cytotoxic T cells is time-consuming and often unsuccessful. An alternative approach involves genetically modifying T cells to express tumor antigen-specific T-cell receptors (TCRs) or chimeric antigen receptors (CARs), enabling the redirection of large numbers of immune cells to target malignant cells. CARs combine the specificity of antibody-derived single-chain variable fragments with T-cell signaling domains (e.g., CD3ζ, CD28, 4-1BB), enabling T cells to recognize and kill antigen-positive cancer cells in an MHC-independent manner. This chapter outlines protocols for large-scale T-cell expansion and medium-scale production of messenger RNA (mRNA) CAR-T cells using electroporation as a delivery method. Two CARs are used as model systems: one targeting CD19 and another targeting an antigen expressed on hematological malignancies and solid tumors.