CRISPR/Cas9-medaited knockout of endogenous T-cell receptor in Jurkat cells and generation of NY-ESO-1-specific T cells: An in vitro study.

Adoptive transfer of T-cell receptor (TCR)-engineered T cells has been successful in mediating favorable clinical outcomes. TCR-engineered T cells can be applied for targeting cancers whose associated antigens are intracellular and presented through major histocompatibility complexes (MHC). The mispairing of the exogenous TCR chains with the endogenous TCR chains leads to functionally impaired TCR-engineered T cells. The CRISPR/Cas9 genome-editing system can be utilized for the knockout of the endogenous TCR in T cells before introducing the exogenous TCR chains. In this study, we used the lentiviral delivery of CRISPR/Cas9 for disrupting the expression of the endogenous TCR in the Jurkat cell line. Next, an exogenous TCR targeting human leukocyte antigen (HLA)-A*0201-restricted New York esophageal squamous cell carcinoma 1 (NY-ESO-1) peptide was transduced into the TCR-knockout (KO) Jurkat cells. Further, we assessed lentiviral transduction efficacy using tetramer assay and evaluated the functionality of the NY-ESO-1-specific TCR-engineered T cells by quantifying the cell surface expression of CD69 upon co-cultivation with peptide-pulsed T2 cells. We successfully knocked out the endogenous TCR in ∼40% of the Jurkat cells. TCR-KO cells were selected and subjected to express NY-ESO-1-specific TCRs using lentiviral vectors. Flow cytometry analysis confirmed that up to 55% of the cells expressed the transgenic TCR on their surface. The functionality assay demonstrated that >90% of the engineered cells expressed CD69 when co-cultured with peptide-pulsed T2 cells. Conclusively, we developed a pipeline to engineer Jurkat cells using the state-of-the-art technique CRISPR/Cas9 and generated TCR-engineered cells that can become activated by a tumor-specific antigen.