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

Abstract

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.

CD44: A Multifunctional Mediator of Cancer Progression

“…The antitumour activity of CD44-CAR T cells was also investigated for hepatocellular carcinoma in vitro and in vivo. CD44-CAR T cells had stronger tumour growth suppression capacity and prolonged survival in CD44 + hepatocellular carcinoma xenograft mice compared to normal and mock T cells [ 231 ]. The constructed bispecific tumour-targeted T cell engager (BiTE) molecule specific for CD44v6 was incorporated into an oncolytic helper binary adenovirus (CAdDuo) encoding an immune checkpoint blocker (PD-L1Ab) and immunostimulatory cytokine (interleukin [IL]-12) to form CAdTrio.…”

c-Met specific CAR-T cells as a targeted therapy for non-small cell lung cancer cell A549

“…On the 28th day, the mice were euthanized by cervical dislocation, followed by removal of the liver, spleen, lung, and kidney tissues of the nude mice, and then overnight fixing was carried out by using paraformaldehyde (4%). After that, paraffin-embedded sections were performed, and H&E staining [ 32 ] was performed. In vitro, the tumor tissues were apoptosis-associated Tunel staining and immunohistochemically stained with proliferation-associated antigen Ki67 to evaluate the effect of c-Met CAR-T cells on the progression of established xenograft tumors [ 25 ].…”

Cellular based immunotherapy for primary liver cancer

“…Intratumoral medication of AFP-CAR-T was reported to lyse HCC cells via cytokine dependent manner and suppress tumor growth in mouse model [ 84 ]. Analogously, CD44-CAR-T [ 85 ] and EGFRvIII-CAR-T [ 86 ] both released higher levels of cytokines such as INF-γ, TNF-α and better suppressed HCC growth compared with normal/mock T group in vitro and vivo . In a phase II clinical trial, patients with advanced HCC received CD133-CAR-T cell infusion after prior systemic therapy, and emerged with 12 months median overall survival (OS) and 6.8 months progression free survival (PFS) [ 71 ].…”