Treating cancers using allogeneic universal CAR T cells resistant to PD1 inhibition generated by multiplex genome editing via CRISPR/Cas9 Supplementary Figure 1. Strategy of introducing CRISPR/Cas9 into T cells. Supplementary Figure 2. Multiple deliveries of gRNAs disrupts genes in human primary T cells with high efficiency without impairing effector function. Supplementary Figure 3. Proliferation test of CD3neg T cells. Supplementary Figure 4. Reduced alloreactivity of TCR and B2M double-disrupted T cells. Supplementary Figure 5. GVHD effect of gene-modified CAR T cells Supplementary Figure 5. Generation of universal CART cells deficient of PD1. Supplementary table 1
ARTICLE 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.
