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Updated: Aug 2, 2025
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced
Elaine Lau1, George Kwong1, Tristan W Fowler1
1Caribou Biosciences, Inc., Berkeley, California, USA.
Background Aims:
Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed 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-generation 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 orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without 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 xenografts 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 exhaustion 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 provide a compelling option for treating B lymphoid malignancies.
Insights
Genomic editing of T cells to disrupt the programmed death-1 (PD-1) checkpoint enhanced CAR-T cell therapy, leading to improved survival in a B cell malignancy model. This approach boosts T cell fitness and anti-tumor activity.
Area of Science:
- Immunotherapy
- Cancer Biology
- Gene Editing
Background:
- Immune checkpoint inhibitors have advanced cancer treatment.
- The PD-1/PD-L1 axis regulates T cell function and can be targeted to enhance CAR-T cell therapy.
Purpose of the Study:
- To investigate the efficacy of genome editing to disrupt the PD-1 checkpoint in allogeneic CAR-T cells.
- To assess the impact of PD-1 knockout on CAR-T cell function and anti-tumor activity in a B cell malignancy model.
Main Methods:
- Primary human T cells were engineered using CRISPR-Cas9 to knock out the PDCD1 gene (encoding PD-1).
- A CD19-specific chimeric antigen receptor (CAR) was inserted into the T cell receptor alpha constant locus.
- Allogeneic CAR-T cells with and without PD-1 knockout (CB-010) were tested in an orthotopic B cell malignancy xenograft model.
Main Results:
- CB-010 cells demonstrated significantly longer survival in tumor-bearing mice compared to CAR-T cells without PD-1 knockout.
- Single-cell RNA-Seq revealed CB-010 cells had reduced T regulatory cells, lower exhaustion/dysfunction, and enhanced activation and metabolic fitness (glycolysis, oxidative phosphorylation).
- Mitochondrial respiration capacity was increased in CB-010 cells, indicating a less differentiated T cell phenotype.
Conclusions:
- Genomic disruption of the PD-1 checkpoint enhances allogeneic CAR-T cell therapy.
- This strategy shows promise for treating B lymphoid malignancies by improving T cell fitness and anti-tumor efficacy.
