Novel mRNA-Engineered Fully Human CAR-T Cells Targeting AXL in Solid Tumors

Bo Zou; Mengge Wang; Shimeng Bai; Ning Li; Zhongyi Fan; Yuanzheng Peng; Mingshu Han; Chen Zeng; Hongzhou Lu; Lin Qi; Xingding Zhang; Xiaohua Tan; Qibin Liao

doi:10.3390/biomedicines13040844

Novel mRNA-Engineered Fully Human CAR-T Cells Targeting AXL in Solid Tumors

Biomedicines. 2025 Apr 1;13(4):844. doi: 10.3390/biomedicines13040844.

Authors

Bo Zou^{1

2}, Mengge Wang³, Shimeng Bai², Ning Li², Zhongyi Fan², Yuanzheng Peng⁴, Mingshu Han², Chen Zeng³, Hongzhou Lu⁵, Lin Qi¹, Xingding Zhang¹, Xiaohua Tan², Qibin Liao²

Affiliations

¹ Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, School of Medicine, Sun Yat-sen University, Shenzhen Campus, Shenzhen 518106, China.
² Biotherapy Clinical Research Center, Shenzhen Third People's Hospital, The Second Affiliated Hospital to Southern University of Science and Technology, Shenzhen 518112, China.
³ Institute of Advanced Biotechnology and School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China.
⁴ Kidney Transplant Department (Liver Transplant Department), Transplant Center, National Clinical Research Center for Infectious Disease, Shenzhen Third People's Hospital, The Second Affiliated Hospital to Southern University of Science and Technology, Shenzhen 518112, China.
⁵ National Clinical Research Centre for Infectious Diseases, Shenzhen Third People's Hospital, The Second Affiliated Hospital to Southern University of Science and Technology, Shenzhen 518112, China.

Abstract

Background/Objectives: The AXL receptor tyrosine kinase is a promising therapeutic target in solid tumors, yet conventional viral vector-engineered CAR-T cells face critical limitations, including risks of insertional mutagenesis and immunogenicity from murine-derived single-chain variable fragments (scFvs). This study aimed to develop and evaluate mRNA-engineered fully human AXL CAR-T (^mfhAXL CAR-T) cells as a safer, scalable alternative for solid tumor immunotherapy. Methods:^mfhAXL CAR-T cells were generated via electroporation-mediated delivery of in vitro transcribed mRNA encoding a fully human AXL-specific CAR. CAR expression kinetics and T-cell viability were quantified by flow cytometry. Antitumor activity was assessed through in vitro co-cultures with AXL-positive lung and pancreatic cancer cells, measuring cytotoxicity, cytokine secretion, and specificity. In vivo efficacy was evaluated in a lung cancer xenograft mouse model, with tumor volume and body weight monitored over 14 days. Results: Flow cytometry confirmed transient but high CAR expression (>90% at 24 h) with preserved T-cell viability (>90%). In vitro, ^mfhAXL CAR-T cells exhibited dose-dependent cytotoxicity and antigen-specific cytokine secretion. In vivo, four administrations of ^mfhAXL CAR-T cells suppressed tumor growth without body weight loss. Conclusions: The mRNA-electroporated ^mfhAXL CAR-T platform enables cost-effective, large-scale production, offering a safer alternative to viral vector-based approaches by eliminating risks of insertional mutagenesis and immunogenicity.

Keywords: AXL; fully human CAR-T; immunotherapy; mRNA; solid tumors.

Abstract

Grants and funding