Targeting Mitophagy Enhances Cytotoxicity of Autologously-Derived CAR-T Cells
Authors: Chen Rongrong , Zhang Mingming , Cen Xufeng , Jin Chunxiang , Luo Yi , Xia Hongguang , Pan Rongqing , Huang He , Hu Yongxian 
Published: December 2024

Introduction

Chimeric antigen receptor (CAR) T cells yield remarkable responses in a variety of hematological malignancies, including relapsed/refractory multiple myeloma (R/RMM) and acute B-cell lymphoblastic leukemia (B-ALL). However, acquired dysfunction of T cell significantly contributes to the failure of autologous CAR-T cell therapy, impeding both longevity and efficacy of CAR-T cells in vivo.

Methods

To investigate the determinants of the dysfunction, an integrated single-cell and bulk RNA sequencing analysis of anti-BCMA CAR-T cells was carried out, involving 26 R/RMM patients and 7 age-matched healthy donors (HD). We hypothesize that the manufacture from defective T cells to CAR-T cells could be reversed by pharmacological or genetic strategies, potentially altering the fate of CAR-T cells.

Results

Transcriptional profiling revealed that defective mitochondrial function is generally present in patient-derived CAR-T cells compared to those from HD. Mitophagy, a central process for maintaining mitochondrial homeostasis, is typically induced to remove damaged mitochondria. Interestingly, dysregulated mitophagy appears to be implicated in CAR-T cells that fail to achieve complete response (CR), exhibiting a simultaneous downregulation of oxidative phosphorylation pathway (P＜0.0001) and the PINK1_PRKN-mediated mitophagy pathway (P=0.0038) compared to CAR-T cells from CR patients. We also verified that exhausted T cells in patients with R/RMM and B-ALL accumulate depolarized mitochondria, showing a significantly lower ratio of tetramethyl rhodamine ethyl ester (TMRE) to MitoTracker Green (MG) than HD (P＜0.0001), which is an indication of mitochondrial activity per mitochondrial mass.

The manufacture of CAR-T cells necessitates rigorous mitochondrial quality control. We discovered that targeting mitophagy of CAR-T cells can restore mitochondrial function. CAR-T cells manufactured with TJ0113 (Tj-CART cells), a novel mitophagy inducer, exhibited increased mitochondrial membrane potential (MMP) and lower reactive oxygen species (ROS). Tj-CART cells also showed an expanded percentage of T memory stem cells production along with elevated TCF1 (P=0.0006) and CCR7 (P=0.0462) expression. Reinforcing mitophagy has been demonstrated to enhance the anti-tumor function and longer persistence of CD19-28ζ and BCMA-41BB CAR-T cells, both in vitro and in vivo. Tj-CART cells upregulate genes such TCF7, MCL-1, BCL2L1 and so on. Mechanistically, TJ0113 restore CAR-T cell function through recruiting Parkin to damage mitochondrial and inducing mitophagy, thereby leading to mitochondrial metabolic remodeling and potentially driving CAR-T cell stemness decisions via the Wnt/β-catenin pathway.

Conclusions

Thus, our results reveal that targeting mitophagy is a tangible pathway to optimize autologously-derived CAR-T cells with mitochondrial dysfunction, providing crucial insights into the how mitophagy regulates stemness and orchestrates mitochondrial metabolism.