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

Abstract

Purpose Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative to 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 the lentiviral delivery of CAR and CRISPR RNA electroporation to co-introduce RNA encoding the Cas9 and gRNAs targeting endogenous TCR, beta-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 anti-tumor 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 anti-tumor activity of the gene-disrupted CART 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.

Is autoimmunity the Achilles' heel of cancer immunotherapy?

“…Epigenetic and transcriptional mechanisms determine the functional plasticity of T cells to switch between their exhausted and effector states 62 , and so altering the transcriptional landscape-for example, by manipulating transcription-factor and gene-enhancer expression-and epigenetic landscape-for example, by manipulating the activity of histone-modifying methylation or demethylation enzymes, histone acetylases, and DNA demethylases-of adoptively transferred T cells may yield a more specific anti-tumor response than generalized PD-1 and CTLA-4 blockade. Genomic editing of chimeric antigen receptor (CAR) T cells to render them resistant to exhaustion has been proposed 63,64 , and Sen and colleagues suggested that mapping state-specific enhancers in exhausted T cells could enable more precise genome editing for adoptive T cell therapy 62 .…”

“…Genome editing to create CAR T cells or TCR transgenic T cells devoid of the endogenous TCR in order to prevent GVHD and enhance the function of the transgenic TCR has been reported 64,100,101 .…”

“…These attempts to create checkpoint-resistant T cells are likely to increase both their general efficacy and autoantigen-mediated effects 63,64,102 . Thus, controlling potential off-target effects of CAR and TCR-transduced T cells will depend on choosing an appropriate tumor-specific antigen, eliminating the endogenous TCR, engineering CAR T cells reliant on multiple tumor-associated antigens for activation 103 , and inserting suicide genes, such as iCaspase-9 or other regulated receptors 75 , that allow for quick inactivation in the event of off-target effects.…”

Low PD-1 Expression in Cytotoxic CD8+ Tumor-Infiltrating Lymphocytes Confers an Immune-Privileged Tissue Microenvironment in NSCLC with a Prognostic and Predictive Value

“…Overall, these findings underline the need to define which subpopulation of TILs expresses PD-1 and whether and to which extent PD-1 expression reflects activation or inhibition. Finally, we are encouraged to support PD-1-to-CD8 ratio as a potential biomarker by the first approved clinical trial applying the CRISPRcas technology in lung cancer, in which PD-1 inhibitory receptor has been genetically disrupted in adoptive cell transfer (43).…”

Emerging applications of gene edited T cells for the treatment of leukemia

“…Cellular repair at such sites by Non Homologous End Joining (NHEJ) creates a variety of insertions and deletions (Indels), disrupting native sequences and, depending on the target site, resulting in defective or absent gene expression. The addition of Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 reagents to the editing toolbox has further extended the range of genomic sites susceptible to DNA scission [15][16][17]. Improvements in the ability to deliver editing reagents into cells as RNA have been a key advance that has allowed translation and scalability to therapeutic strategies.…”