Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition

TLDR

The CRISPR/Cas9 system has emerged as a simple and efficient method for multiplex genome engineering. The study aims to use gene‑disrupted allogeneic T cells as universal effector cells to improve CAR T‑cell therapy for cancers and infectious diseases. The authors combined lentiviral CAR delivery with electroporation of Cas9 mRNA and gRNAs targeting TCR, β‑2 microglobulin, and PD1 to generate allogeneic CAR T cells lacking TCR, HLA‑I, and PD1. The edited CAR T cells showed potent antitumor activity comparable to non‑edited cells, reduced alloreactivity without graft‑versus‑host disease, and triple editing including PD1 further enhanced in vivo efficacy, indicating their potential as universal donor effector cells against cancers and infectious diseases. Published in Clinical Cancer Research 23(9):2255–66, ©2016 AACR.

Abstract

Abstract Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases. Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1. Results: The CRISPR gene–edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene–edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells. Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 23(9); 2255–66. ©2016 AACR.