TGF-ß inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors

Tang N, Cheng C, Zhang X, Qiao M, Li N, Mu W, Wei XF, Han W, Wang H.
Publication Date: (2020)
Issue: 27;5(4): e133977
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
Hep G2
Species: human
Tissue Origin: liver
Culture Media:
4D-Nucleofector® X-Unit

6 µg Cas9 protein (provided by Shenzhen Fapon Biological Therapy Co. Ltd.) with 6 µg sgRNA was incubated at room temperature for 20 minutes. 5 × 10^5 HepG2 or CAR T cells were centrifuged at 200 g for 5 minutes, resuspended in 20 µl transfection buffer containing indicated ribonucleoprotein, and then transferred into the electroporation cuvette. 4D-Nucleofector System N (Lonza), SE cell line 4D-nucleofector kit (V4XC-1032, Lonza), and program EH-100 were used for HepG2 cell line electroporation. 4D-Nucleofector System N (Lonza), P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza), and program EO-115 were used for CAR T cell electroporation. 


In recent years, chimeric antigen receptor-modified T cell (CAR T cell) therapy has proven to be a promising approach against cancer. Nonetheless, this approach still faces multiple challenges in eliminating solid tumors, one of which being the immunosuppressive tumor microenvironment (TME). Here, we demonstrated that knocking out the endogenous TGF-ß receptor II (TGFBR2) in CAR T cells with CRISPR/Cas9 technology could reduce the induced Treg conversion and prevent the exhaustion of CAR T ce lls. Meanwhile, TGFBR2-edited CAR T cells had better in vivo tumor elimination efficacy, both in cell line-derived xenograft and patient-derived xenograft solid tumor models, whether administered locally or systemically. In addition, the TGFBR2-edited CAR T cells could eliminate contralaterally reinoculated xenografts in mice effectively, with an increased proportion of memory subsets within circulating CAR T cells of central memory and effector memory subsets. In conclusion, we greatly improved the in vitro and in vivo function of CAR T cells in TGF-ß-rich tumor environments by knocking out endogenous TGFBR2 and propose a potentially new method to improve the efficacy of CAR T cell therapy for treating solid tumors.