mRNA-based AXL-targeting chimeric antigen receptor-T cells against solid tumors

Abstract Background Currently approved CAR-T cells primarily utilize retroviral or lentiviral vectors (LV), posing significant challenges due to their time-consuming and costly nature, and associated safety concerns, including insertional mutagenesis. Consequently, mRNA delivery holds promising prospects in CAR-T cell engineering. AXL represents an ideal target for LV-engineered CAR-T cells in the treatment of various malignancies, including breast, lung, and pancreatic cancer. However, the anti-tumor activity of mRNA-based AXL CAR-T cells remains to be fully elucidated. Methods We developed a novel CAR utilizing a fully humanized AXL-specific antibody. Subsequently, the mRNA encoding AXL-specific CAR was synthesized via in vitro transcription. This mRNA was then transfected into pre-activated T cells through electroporation. The expression levels of mRNA-based CAR were evaluated by flow cytometry. Furthermore, in vitro and in vivo assays were performed to evaluate the antigen-specific cytotoxicity and cytokine release capabilities of mRNA-based AXL CAR-T cells in various tumor cells and in a tumor-bearing mouse model. Results Electroporation of mRNA-encoding AXL CAR minimally affected T cell viability, resulting in high CAR expression over 90%, and this CAR expression persisted for at least three days following electroporation, consistent with observed cytotoxicity against the lung cancer cell line A549. mRNA-based AXL CAR-T cells exhibited dose-dependent cytotoxicity against both A549 and pancreatic cancer cell line Panc-1, achieving a killing rate exceeding 80% at an effector:target ratio of 4:1. Additionally, mRNA-based AXL CAR-T cells specifically secreted high level of cytokines IL-2 and IFN-γ upon interaction with target tumor cells. mRNA-based AXL CAR-T cells exhibited minimal impact on T cell subtypes while demonstrating robust cytotoxicity comparable to LV-engineered CAR-T cells. Additionally, mRNA-based AXL CAR-T cells demonstrated good anti-tumor activity in a subcutaneous lung cancer xenograft model. Conclusions mRNA-based AXL CAR-T cells transiently expressed high levels of CAR, enabling cost-effective large-scale production, and comparable functionality to LV-engineered CAR-T cells within a short time frame, which offers a safe alternative for clinical therapy utilizing LV-engineered CAR-T cells. mRNA-based AXL CAR-T cells have shown promising results in treating solid tumors, including lung cancer and pancreatic cancer, establishing an experimental basis for their potential clinical application in solid tumors.