Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness

Background aims: Therapeutic disruption of immune checkpoints has significantly advanced the armamen-tarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/pro-grammed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy.Methods: To address checkpoint interference, primary human T cells were genome edited with a next -gener-ation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity.Results: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established ortho-topic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells with-out a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xeno-grafts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaus-tion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells.Conclusions: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may pro-vide a compelling option for treating B lymphoid malignancies. & COPY; 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Automated summary

This study explores the potential of combining PD-1 blockade with CAR-T cell therapy for treating B cell malignancies, by genetically editing T cells to disrupt the PD-1 checkpoint.