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

Program: Oral and Poster Abstracts
Type: Oral
Session: 703. Cellular Immunotherapies: Basic and Translational III
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Therapies, metabolism, Biological Processes
Monday, December 12, 2022: 4:45 PM

Shinpei Harada1*, Daigo Hashimoto, MD2, Hajime Senjo, M.D.2, Kazuki Yoneda, M.D.2*, Zixuan Zhang2*, Xuanzhong Chen2*, Ryo Kikuchi, M.D.2*, Masahiro Chiba, M.D.2*, Hiroyuki Ohigashi, M.D., Ph.D.2*, Takahide Ara, M.D., Ph.D.2*, Masao Nakagawa, M.D., Ph.D.2, Masashi Suganuma, M.D., Ph.D.3*, Rick C. Tsai, M.D., D.M.D.3* and Takanori Teshima, M.D., Ph.D.2

1Department of Hematology, Hokkaido University Faculty of Medicine, Sapporo City, Japan
2Department of Hematology, Hokkaido University Faculty of Medicine, Sapporo, Japan
3LUCA Science Inc., Tokyo, Japan

[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.

Disclosures: Suganuma: Luca Science Inc: Current Employment. Teshima: Eisai: Research Funding; NIPPON SHINYAKU: Research Funding; Fuji Pharma: Research Funding; Kyowa Kirin: Honoraria, Research Funding; Astellas: Research Funding; Chugai: Research Funding; Bristol-Myers Squibb: Honoraria; Asahi Kasei Pharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding; Janssen: Other: Manuscript preparation; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Manuscript preparation; Luca Science Inc.: Research Funding; Sumitomo Pharma: Research Funding; ONO: Research Funding; SHIONOGI: Research Funding; Priothera SAS: Research Funding; Otsuka: Research Funding; AbbVie: Honoraria; Celgene: Honoraria; Meiji Seika Pharma: Other: non-financial support; DAIICHI SANKYO: Other: non-financial support ; AstraZeneca: Other: non-financial support ; Roche Diagnostics: Other: non-financial support .

*signifies non-member of ASH