Engineering of induced pluripotent stem cells for the efficient development of non-alloreactive, hypoimmunogenic CD8αβ CAR-T cells

Background: Induced pluripotent stem cells (iPSCs) are a promising platform to produce "off-the-shelf" chimeric antigen receptor (CAR)-engineered T cells (CAR-T) with thepotential for multiplex genetic engineering. Methods: Here, we employed genome editing to knock out the TRAC and B2M genes in iPSC lines, while simultaneously harnessing the edited loci to introduce a drug-inducible CAR and the human leukocyte antigen (HLA)-E single chain trimer. Results: The inducible CAR expression allowed the robust generation of T-cell receptor (TCR)-negative CD8ab+ CAR-T cells with demonstrable anti-tumor efficacy and lack of alloreactivity. HLA-class Inegative, HLA-Epositive CD8 CAR-T cells were protected against immune rejection; however, disruption of B2M resulted in genomic instability and affected the efficiency of T-cell development and the functionality of the generated T cells. Discussion: Facilitating multiplex engineering at well-characterized genomic loci and regulation of possible developmental effects will empower the use of engineered iPSC as a viable method to efficiently produce "off-the-shelf" CART cells.