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

## TL;DR

Scientists engineered stem cells to create CAR-T cells that avoid immune rejection and work against tumors.

## Contribution

A novel method for generating hypoimmunogenic, non-alloreactive CD8αβ CAR-T cells using genome-edited iPSCs.

## Key findings

- Genome-edited iPSCs produced TCR-negative CD8αβ CAR-T cells with anti-tumor activity and no alloreactivity.
- HLA-E expression protected CAR-T cells from immune rejection, but B2M disruption caused genomic instability.
- Multiplex engineering at specific loci improves iPSC-based CAR-T cell development efficiency.

## Abstract

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.

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.

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.

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.