Intercellular Mitochondrial Transfer Enhances Metabolic Fitness and Anti-Tumor Effects of CAR T Cells

[Introduction] Chimeric antigen receptor (CAR) T-cell therapy is a potentially curative treatment for patients with relapsed or refractory (r/r) hematopoietic malignancies. However, roughly half of patients with r/r B-cell lymphoma experience treatment failure after CAR T-cell therapy possibly due to poor expansion, limited persistence, and exhaustion of CAR T cells. Given that metabolic fitness has a central role in regulating T-cell functions, manipulating the mitochondrial function is a promising approach to enhance anti-tumor effects of CAR T cells. In the current study, we tested if transfer of mitochondria isolated from HeLa cells using a novel intact mitochondrial isolation technology (Q mitochondria; WO2021015298 A1) into T cells could enhance metabolic fitness and anti-tumor effects of CAR T cells. [Methods] Purified T cells from naïve mice were stimulated with plate-bound anti-CD3/CD28 antibodies in the presence or absence of Q (50-100 µg/ml). Culture medium and Q were renewed every 24 or 48 hours. Mitochondrial respiratory function was evaluated using the XFp Flux Analyzer. For CAR T-cell generation, purified T cells were stimulated with anti-CD3/-CD28 antibodies for 48 hrs and then incubated with retroviral vector encoding anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hrs. [Results] First, we confirmed that mitochondrial structure and ATP synthesis of Q mitochondria were much better maintained compared to those of mitochondria prepared by the conventional homogenization method. When purified T cells were incubated with fluorescent-labeled Q mitochondria for 24 hrs during TCR stimulation, approx. 95% of T cells endocytosed Q mitochondria, and mitochondrial mass per cell was significantly increased. As expected, oxidative phosphorylation (OXPHOS) was markedly enhanced in Q-treated T cells compared to diluent-treated controls after TCR stimulation. The levels of reactive oxygen species (ROS) in diluent-treated T cells were increased after 72-hrs TCR stimulation, while Q mitochondria significantly mitigated accumulation of ROS in activated T cells. Q mitochondria increased TCF-1⁺PD-1⁺Tim3^- precursor exhausted T cells and enhanced production of IFN-g and TNF-a after 72-hrs TCR stimulation, and improved survival after 192-hrs chronic TCR stimulation. Based on these findings, we next investigated whether Q mitochondria could affect CAR T-cell functions. CAR T cells were generated with purified T cells from naïve BALB/c mice and Q mitochondria was added from the beginning of TCR stimulation. Q-treated CAR T cells demonstrated significantly enhanced OXPHOS (Fig. 1) and cytokine production compared to diluent-treated CAR T cells. Q-treated CAR T cells also showed significantly improved proliferative capacity and in vitro cytotoxicity against syngeneic B-cell lymphoma cells (A20). Next, we examined in vivo antitumor effects of Q-treated CAR T cells. Naïve BALB/c mice were subcutaneously injected with 1.5 x 10⁷ A20 cells on the right flank and intravenously injected with 1 x 10⁶ Q-treated or control CAR T cells, or naïve BALB/c T cells 14 days after tumor inoculation. Although control CAR T cells significantly suppressed tumor growth compared to naïve T cells, all mice died due to tumor growth by day 31 after tumor inoculation. Importantly, tumor growth was significantly delayed (Fig. 2) in the mice injected with Q-treated CAR T cells leading to significantly prolonged survival compared to those treated with control CAR-T cells. [Conclusions] We for the first time found transfer of Q mitochondria prepared with the intact mitochondrial isolation technology into CAR T cells improved metabolic fitness, leading to enhanced proliferative capacity and cytokine production, and in vitro and in vivo anti-tumor effects. Transfer of Q mitochondria could be also useful as an adjunctive method in combination with various other genetical and pharmacological approaches to enhance effectiveness of CAR T-cell therapy.

Fig. 1. Oxygen consumption rate (OCR) of Q-treated and control CAR T cells. *, P < 0.05; **, P < 0.01.

Fig. 2. Tumor growth curves in the mice injected with Q-treated CAR T cells, control CAR T cells, and naïve T cells on day 14 after tumor inoculation. *, P < 0.05; **, P < 0.01.