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

Abstract

Hezhi Wang,1 Xueshuai Ye,1,2 Yi Ju,3 Ziqi Cai,2 Xiaoxiao Wang,4 Pingping Du,2 Mengya Zhang,2 Yang Li,1 Jianhui Cai1,5 1Department of Surgery, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Hebei Engineering Technology Research Center for Cell Therapy, Hebei HOFOY Biotech Corporation Ltd., Shijiazhuang, Hebei, People’s Republic of China; 3Department of Medicine, Medical College of Hebei University of Engineering, Handan, Hebei, People’s Republic of China; 4Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 5Department of Surgery & Oncology, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Jianhui CaiDepartment of Surgery & Oncology, Hebei General Hospital, 348 West Heping Road, Shijiazhuang, Hebei 050051, People’s Republic of ChinaTel [phone]Fax [phone]Email [email protected]: 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.Keywords: cancer immunotherapy, chimeric antigen receptor, nonviral vector, cancer stem cell antigen, tumor suppression

Keywords

• cancer immunotherapy
• chimeric antigen receptor
• nonviral vector
• cancer stem cell antigen
• tumor suppression