CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells.

Zhang Y, Zhang X, Cheng C, Mu W, Liu X, Li N, Wei X, Liu X, Xia C, Wang H.
Source: Frontiers in Immunology
Publication Date: (2017)
Issue: 1: 1-9
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
T cell, human cord blood unstim.
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit
T cells were stimulated for 3 days, and then 1  106 cellswere electroporated with 3 mg Cas9 protein (Thermo Fisher Scientific) and 3 mg sgRNAs targeting the LAG-3 exon1 by 4D-Nucleofector System N (Lonza) using the P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza) according to manufacturer instructions. Program EO-115 was used.
T cells engineered with chimeric antigen receptor (CAR) have been successfully applied to treat advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of Tcell activity is lymphocyte activation gene-3 (LAG-3). We successfully generated LAG-3 knockout Tand CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.