Minicircle DNA-Mediated CAR T Cells Targeting CD44 Suppressed Hepatocellular Carcinoma Both in vitro and in vivo.

Purpose: Based on the continuous exploration of solid tumor immunotherapy, we focused on hepatocellular carcinoma with a high level of morbidity and mortality. We confirm the stability of mcDNA-based CAR T cell generating platform, and investigate the antitumor activity of CD44-CAR T cells against hepatocellular carcinoma both in vitro and in vivo. Materials and Methods: We fused anti-CD44 scFv structure with transmembrane domain and intracellular domain Using a non-viral mcDNA vector to load CD44-CAR gene, then transfected the mcDNA-CD44-CAR into human T cells by electroporation. We exhibited the transfection efficacy of CAR T cells and the CD44 expression of tumor cell lines by flow cytometry. The antitumor efficacy of CD44-CAR T cells in vitro and in vivo was detected through CCK-8 and ELISA assays, and xenograft mouse models, respectively. Results: We obtained mcDNA-CD44-CAR with a high level of density after repeated extraction and purification. The expression efficacy of CD44-CAR in T cells was more than 50% after seven days electroporation and the phenotype of CD44-CAR T cells was no difference compared with normal T cells. For CD44-positive hepatocellular carcinoma xenograft mice, CD44-CAR T cells had stronger tumor growth suppression compared to normal T and mock T cells. The same results occurred on the in vitro experiments including cytokine secretion and cytotoxicity assays. H&E staining graphs revealed that CD44-CAR T cells did not induce side effects in xenograft mice. Conclusion: The strategy for generating CAR T cells targeting cancer stem cell antigens was efficient and concise. The mcDNA had superior transgene ability without virus-related adverse effects. CD44-CAR T cells had strong suppression capacity against hepatocellular carcinoma.