CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells

Abstract

Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies.

Chimeric Antigen Receptor T Cells: Extending Translation from Liquid to Solid Tumors

“…For example, engineering CARs with different costimulatory or signaling domains may be an alternative to PD-1 ablation. 114 Interestingly, CAR T cells designed to express a PD-1-CD28 switch receptor, in which the extracellular domain of PD-1 is fused to the CD28 transmembrane and signaling domains, exhibited enhanced activity against solid tumors (Fig. 1).…”

“…Nucleofection of CRISPR-Cas9 ribonucleoprotein to disrupt PD-1 in CAR T cells resulted in anti-CD19 CAR T cells with better tumor control in vitro and in an in vivo murine subcutaneous tumor xenograft model, raising the possibility that CAR T-cell activity can be improved by exploiting the mechanisms through which PD-1/PD-L1 signaling inhibit CAR T-cell function or signaling. 114 Several studies have explored the possibility of using CRISPR-Cas9 for multiplex genome editing to generate CAR T cells with improved function. T-cell receptor a chain (TRAC) and beta-2 microglobulin (B2M, essential for HLA-I heterodimer cell surface expression) were targeted to create double-knockout CAR T cells, while TRAC, B2M, and PD-1 were disrupted to make triple knockout CAR T cells.…”

Off the shelf T cell therapies for hematologic malignancies

“…CAR-T cells containing multiple gene edits that include knockout of inhibitory signaling receptors such as PD-1 (PDCD1) [29,30], LAG-3 [31], or knock-in of a dominant negative TGFβ2R (TGFBR2) [32] have been described and are expected to enter into clinical trials in the near future.…”

T lymphocytes as therapeutic arsenal for patients with hematological malignancies

“…In an attempt to improve safety and potency, advances in of CRISPR/Cas9 [130] and TALEN-mediated [131] genome-editing approaches for the large-scale manufacturing of 'off-the-shelf' CAR-T cells are outperforming conventionally transduction methods (NCT03399448). The genetically modify Sleeping Beauty transposon/transposase system to accelerate and increase the availability of CARs, has been also evaluated on a phase I trial [132].…”