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

Alexandros Nianias^1,2Afroditi Katsarou^1,2Henk-Jan Prins^1,2Aida Shahrabi¹Cindy van Velzen^1,2Linda Henneman³Ruud Ruiter^1,2Tuna Mutis^1,2Richard Groen^1,2Maria Themeli^1,2*

• ¹Hematology, Amsterdam UMC Locatie VUmc, Amsterdam, Netherlands
• ²Cancer Biology and Immunology, cancer center amsterdam, Amsterdam, Netherlands
• ³Animal Modeling Facility, Netherlands Cancer Institute, Amsterdam, Netherlands

Induced pluripotent stem cells (iPSC) is a promising platform to produce "off-the-shelf" chimeric antigen receptor (CAR)-engineered T cells (CAR-T) with the potential 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 CD8αβ+ 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" CAR-T cells.