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3455 GLUT1 Overexpression Enhances 19-28z CAR-T Cell Expansion and Metabolic Fitness

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Translational Research
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Yuzhe SHI1*, Ivan S Kotchetkov, MD2*, Sophie Alexandra Hanina, BMBCh, PhD2* and Michel Sadelain, MD PhD2

1Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, NEW YORK, NY
2Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY

The tumor microenvironment presents many obstacles to effective CAR T cell therapy. One of these obstacles is glucose competition from tumor and myeloid cells to the detriment of CAR T cells. We hypothesize here that CAR T cells with elevated levels of glucose transporter are better fit under such conditions, which will result in improved therapeutic efficacy.

We overexpressed either GLUT1 or GLUT3 in CD19-targeted CAR T cells engineered with a clinically validated CAR (19-28z). GLUT1 is ubiquitously expressed in tissues and GLUT3 has a higher affinity for glucose. Upon in vitro antigen stimulation, 1928z-GLUT1 and 1928z-GLUT3 showed greater enrichment of CAR+ T cells and underwent more cell divisions than control 1928z-LNGFR under glucose-limiting conditions. In repetitive antigen stimulation assays, 1928z-GLUT1 expanded significantly more than 1928z-LNGFR cells in 10 mM or 5 mM glucose medium. Surprisingly, 1928z-GLUT3 CAR T cells faired markedly less well than 1928z-LNGFR controls, associated with reduced viability of 1928z-GLUT3 CAR T cells after 2 or 3 exposures to antigen. In the NALM6 acute lymphoblastic leukemia model, low-dose CAR T cell treatment with 1928z-LNGFR and 1928z-GLUT3 did not curtail rapid tumor progression, while 1928z-GLUT1 CAR T cells significantly increased survival. More abundant CAR+ T cells were found in the bone marrow and spleen of 1928z-GLUT1-treated mice. Interestingly, we observed consistent increases in CD62L+CD45RA+ Tscm population in 1928z-GLUT1 cells, including both CD8 and CD4 T cells. We flow-sorted CAR T cells from treated mice and performed SMARTer RNA-seq. GLUT1, but not GLUT3, significantly upregulated TCF7 and genes encoding glycolytic enzymes and electron transport chain, indicating that GLUT1 overexpression enhances both glycolysis and oxidative phosphorylation.

To further test GLUT1-CAR-T function in tumor lines with high levels of glucose transporter, we generated a renal cell carcinoma (RCC) patient-derived xenograft (PDX) with ectopic expression of human CD19. CAR-T cells were repetitively stimulated with the RCC PDX under titrated glucose concentrations. 1928z-GLUT1 significantly improved CAR T cell viability compared to 1928z-LNGFR in low glucose conditions. The expansion of GLUT1 CAR-T cells in 10 mM and 5 mM glucose also surpassed that of LNGFR CAR-T cells. In vivo, 1928z-GLUT1-treated mice showed significantly less tumor burden than 1928z-LNGFR-treated mice.

Overall, we developed a novel strategy for enhancing the metabolic fitness of 1928z CAR-T cells by constitutively overexpressing GLUT1. GLUT1 but not GLUT3 enhances glycolysis and oxidative phosphorylation in CAR-T cells, which sustains T-cell expansion and increases therapeutic efficacy. This strategy is under investigation with other CAR formats.

Disclosures: Sadelain: Mnemo: Current equity holder in private company, Research Funding; Fate: Research Funding; Minerva: Current equity holder in private company; Atara: Research Funding; Takeda: Research Funding.

*signifies non-member of ASH