Precision gene-engineering meets cancer immunotherapy: Targeted insertion of chimeric antigen receptors into the T-cell genome using a novel, virus-free CRISPR/Cas9 knock-in strategy

Introduction Immunotherapy with gene-engineered CAR-T-cells is a transformative cancer treatment. In conventional CAR-T-cell products, gene-transfer is accomplished with viral vectors that are associated with genotoxicity, a risk for insertional mutagenesis, and substantial effort and expense of vector production and handling. To overcome these limitations, we established virus-free targeted insertion to direct the CAR-transgene into defined genomic loci in human T-cells.

Results We validated CRISPR gRNAs targeting the endogenous T-cell-receptor (TCR), and generated CAR knock-in T-cells. We confirmed TCR knock-down and CAR knock-in by flow cytometry and on the genomic level. Notably, CAR knock-in T-cells displayed i. lower and more homogeneous CAR surface expression, ii. activation-dependent CAR regulation, and iii. reduced levels of baseline activation and exhaustion marker expression compared to T-cells with random genomic insertion.

CAR knock-in T-cells conferred specific cytotoxicity, cytokine secretion, and outperformed conventional (random insertion) CAR-T-cells in antigen-dependent proliferation. These data were confirmed with several CAR-constructs of relevance in hematologic (CD19) and solid tumor indications (ROR1/2). Preliminary data also suggest that targeted CAR insertion into the TCR locus is advantageous to the PD1 locus, likely because it enables T-cells to regulate CAR expression physiologically, in sync with their activation status. Accordingly, we consistently found improved viability and lower rates of AICD in knock-in CAR-T-cells after a tumor challenge.

Outlook We are presenting virus-free targeted CAR insertion into a genomic safe harbor as a strategy to augment safety and performance of CAR-T-cell therapy; and consider precision genomic engineering as a key enabling technology in the field of ATMPs.