Genetic Modification Strategies to Enhance the Anti-Tumor Efficacy of CAR-T Cells

CAR-T cell therapy has shown significant efficacy in treating hematologic malignancies such as leukemia and lymphoma, prompting research into its application in various solid tumors. Among these tumors, glioblastoma (GBM) stands out as a particularly challenging target due to its aggressive nature and the lack of effective treatment options.

Although early clinical trials have shown promise, outcomes have often fallen short of expectations, primarily due to T cell exhaustion caused by prolonged antigen exposure. T cell exhaustion manifests as decreased proliferative capacity, impaired effector function, reduced cytokine production, and upregulation of multiple inhibitory receptors. Crucially, this exhaustion cannot be completely reversed by immune checkpoint blockade (ICB) therapy, partly due to the immunosuppressive tumor microenvironment within solid tumors.

Recent advances in single-cell RNA sequencing (scRNA-seq) of tumor-infiltrating lymphocytes (TILs) have provided valuable insights into the molecular mechanisms of T cell exhaustion. Genes such as DNMT3A, SOX4, and PRDM1 have been identified as key regulators limiting T cell anti-tumor activity. Knocking out each gene individually enhanced the anti-exhaustion ability of CAR-T cells. This suggests that genetic modification of these exhaustion-related genes may be an effective strategy to overcome tumor microenvironment limitations and improve the efficacy of CAR-T cell therapy.

Recently, researchers published a research paper entitled "Enhanced FOS expression improves tumor clearance and resists exhaustion in NR4A3-deficient CAR T cells under chronic antigen exposure" in Science Advances. This study found that knocking down NR4A3 enhanced the ability of CAR-T cells to fight malignant gliomas, but this ability was weakened by T cell exhaustion induced by continuous antigen exposure. Enhancing FOS expression on this basis reversed the aforementioned T cell functional exhaustion, thereby maintaining the tumor-clearing ability of CAR-T cells and enhancing therapeutic efficacy. This discovery provides new ideas and strategies for optimizing the clinical treatment of CAR-T cells.

Figure 1. FOS OE/NR4A3 KD enhances in vivo CAR T cell antitumor activity by preserving T cells from exhaustion. (Yin P, et al., 2025)

Functional impairment of CAR-T cells in the tumor microenvironment, i.e., T cell exhaustion, is a major obstacle to the poor efficacy of CAR-T cell therapy in solid tumors. Through single-cell RNA sequencing analysis of tumor-infiltrating T cells from glioma patients, the research team discovered that the NR4A gene family is closely related to T cell exhaustion and is co-expressed with the dysfunctional genes HAVCR2 and TIGIT.

Based on this finding, the research team knocked out NR4A1, NR4A2, and NR4A3 in CAR-T cells. The results showed that CAR-T cells with NR4A3 knockdown exhibited stronger cytotoxic activity against tumors, thereby improving tumor clearance and prolonging survival in vivo. However, with continued increase in tumor burden, the anti-exhaustion phenotype of CAR-T cells induced by NR4A3 knockdown gradually weakened. This weakening is associated with compensatory downregulation of FOS induced by chronic antigen exposure after NR4A3 knockdown.

Therefore, the research team further constructed CAR-T cells that overexpressed FOS and knocked down NR4A3. The results showed that this dual modification shifted the CAR-T cell phenotype and transcriptional profile from a depleted state to an enhanced effector function state, thereby significantly enhancing the anti-tumor effect of CAR-T cells. These findings provide a promising new strategy for improving CAR-T cell therapy in clinical practice and enhancing its therapeutic efficacy against solid tumors.

In summary, this study highlights the crucial role of NR4A3 in regulating human T cell cytotoxicity, memory formation, and anti-depletion capabilities in the early stages of antigen exposure. Furthermore, this study proposes a combined gene modification strategy for CAR-T cells-NR4A3 knockdown and FOS overexpression-to achieve sustained clearance of solid tumors by protecting CAR-T cells from depletion.

Reference

Yin P, et al. Enhanced FOS expression improves tumor clearance and resists exhaustion in NR4A3-deficient CAR T cells under chronic antigen exposure. Science Advances, 2025, 11(42): eadw3571.