Abstract 1774: Pre-treatment of CAR-T cells with met+rap improves the mitochondrial function and anti-tumor efficacy in a hypoxic glioma microenvironment
Authors: Hatae Ryusuke , Kyewalabye Keith , Yamamichi Akane , Chen Tiffany , Phyu Su , Chuntova Pavlina , Okada Hideho 
Published: April 2023

Abstract
Chimeric antigen receptor (CAR)-T therapy has not shown significant therapeutic effects on brain tumors. As our data with a syngeneic mouse model of CAR therapy showed significant hypoxia in the brain tumor microenvironment, we sought to develop a novel strategy to improve the metabolic state of the CAR-T cells in the hypoxic tumor microenvironment. In mice bearing intracerebral SB28-EGFRvIII gliomas receiving an intravenous infusion of EGFR vIII-targeting CAR-T cells, we found mitochondrial ATP production in tumor-infiltrating CAR-T cells was significantly impaired compared to those migrating to the spleen. Therefore, we hypothesized that treating CAR-T cells with metabolic regulators that activate mitochondria before infusion would improve the therapeutic effect of CAR-T cells. Using a hypoxic incubator to mimic the tumor microenvironment of brain tumors, we screened drugs that would improve the metabolic state of CAR-T cells and maintain their anti-tumor effect. Although the mammalian target of rapamycin (mTOR) inhibitor rapamycin and AMP-activated protein kinase (AMPK) activator metformin are ineffective when treated as single drugs, a combination of these drugs (met+rap) prolonged the anti-glioma effect of CAR-T cells under hypoxic conditions. Furthermore, Seahorse assays revealed a higher mitochondrial spare respiratory capacity of met+rap-treated CAR-T cells compared to single drug-treated CAR-T cells or those without pre-treatment. A single intravenous infusion of CAR-T cells pre-treated with met+rap significantly prolonged the survival of mice bearing intracerebral SB28-EGRFvIII tumors compared with CAR-T cells without the pre-treatment. In mass cytometric analyses, a significantly higher number of glioma-infiltrating CAR-T cells were found in mice that received met+rap-treated CAR-T cells compared with mice that received control CAR-T cells. Interestingly, the glioma tissues infiltrated by met+rap pre-treated CAR-T cells were infiltrated by significantly fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells. As a major translational significance, because the CAR-T cells were pre-treated with the drugs during the in vitro expansion with no need for in vivo drug administrations, our strategy would circumvent any toxicity associated with in vivo treatments with the same medications.
These data warrant a translational and clinical evaluation of met+rap pre-treated CAR-T cells in human settings and the development of a CAR-T cell trial using this approach.
Citation Format: Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Hideho Okada. Pre-treatment of CAR-T cells with met+rap improves the mitochondrial function and anti-tumor efficacy in a hypoxic glioma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1774.