Abstract 873: 2-deoxy-D-glucose sensitizes the antigen recognition of chimeric antigen receptor-expressing tumor-infiltrating lymphocytes toward hepatocellular carcinoma cells

Abstract Background: Tumor-infiltrating lymphocytes (TILs) are heterogeneous populations of lymphocytes residing in tumor tissues, comprising various T-cell clones with specificity towards tumor or viral antigens. TIL therapy has been recently approved for advanced melanoma treatment with high-dose interleukin-2 (IL-2) as a concomitant treatment. However, the accessibility of tumor tissues, the varied abundance and effectiveness of tumor-reactive T-cells, and the toxicity of IL-2 infusion limit the clinical application of TILs toward a broader range of tumor indications. To address the high individual variance, we have engineered TILs expressing chimeric antigen receptor (CAR) constructs targeting glycoproteins on the hepatocellular carcinoma (HCC) cell surface. However, heavy glycosylation of these glycoproteins may mask peptidic epitopes to single-chain antibodies. Previous studies have revealed that the glucose/mannose analog, 2-deoxy-D-glucose (2-DG) treatment of pancreatic cancer cells can disrupt the synthesis of N-glycan moieties on the cell surface and enhance CAR T-cell activity. 2-DG treatment of T cells augmented antitumor activity and cell surface retention of IL-2R of T cells. In this study, we investigated whether the anti-tumor function of CAR-TILs against HCC lines would also benefit from the 2-DG treatment. Methods: TILs were isolated from fresh or cryopreserved tumor biopsy samples and stimulated with anti-CD3/CD28 antibodies. Thereafter, TILs were lentiviral transduced with Glypican 3 (GPC3) CAR and membrane bound-interleukins, expanded with γ-irradiated feeder cells in an optimized rapid expansion process (REP), and cryopreserved until use. Phenotypic analyses were performed by flow cytometry. HCC lines were pre-treated with or without 2-DG, and the antigen recognition by antibodies targeting GPC3, CD133, and CD44v6 was evaluated by flow cytometry. In vitro effector function was assessed by IFN-γ release (ELISA) and killing assay (Incucyte). The anti-tumor efficacy of CAR-TILs with or without 2-DG treatment was further assessed in the immunocompromised xenograft model. Results: Pre-treatment of HCC cell lines with 2-DG (1∼10 mM) enhanced the antibody recognition of GPC3 in a dose-dependent fashion while showing no improvement in mAb binding towards CD133/CD44v6. CAR-TILs showed enhanced killing and more IFNγ release towards 2-DG pre-treated GPC3+ HCC lines. The synergistic effect of 2-DG with CAR-TILs was further validated in the immunocompromised murine model, and no apparent toxicity was observed. The proliferation and the CD4/CD8 ratio of CAR-TILs were not affected by 2-DG treatment. Conclusion: 2-DG sensitized GPC3 antigen recognition, thus boosting CAR-TIL cytotoxic activity against GPC3+ HCC cells. These data prompt further investigation before pursuing a combination therapy. Citation Format: Mingyu Liu, Deping Han, Jean-Paul Thiery, Xi Zhang. 2-deoxy-D-glucose sensitizes the antigen recognition of chimeric antigen receptor-expressing tumor-infiltrating lymphocytes toward hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 873.