Abstract A021: Investigation of the impact of the HIF-2α inhibitor belzutifan on T-cell cytotoxicity in renal cell carcinoma (RCC)

Abstract Introduction: RCC is characterized by a hypoxic tumor microenvironment that recruits immunosuppressive macrophages and regulatory T cells to suppress anti-tumor immunity. Belzutifan, a hypoxia-inducible factor 2 alpha (HIF-2α) inhibitor is a standard therapy in advanced RCC. Some preclinical murine data suggests that hypoxia signaling is important for effector T-cell function (Doedens, et al. Nat Immunol. 2013; Veliça, et al. Cancer Immunol Res. 2021), raising the concern that pharmacologic HIF-2α inhibition might impair anti-tumor immunity. The impact of belzutifan on human T-cell function, especially cytotoxic activity, is unknown. Therefore, we established an in vitro RCC antigen model system to evaluate the impact of belzutifan on T-cell cytotoxicity. Methods: Cells from the human VHL-mutated RCC cell line 786-O (parental HLA-A*03: 01) were used as target cells. Endogenous HLA class I was knocked out using a CRISPR–Cas9 system, and cells were transduced with a lentiviral construct to express HLA-A*02: 01, CTAG1B (NY-ESO-1), and HIF-2α (wild-type or belzutifan-resistant G323E mutant), yielding 786-O-WT and 786-O-G323E lines. The G323E mutation disrupts belzutifan binding to HIF-2α, so that any differences observed during the killing assay are attributable to the effect of belzutifan on T cells rather than cytotoxic effects of belzutifan on 786-O-G323E RCC cells. Human T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and engineered to express the HLA-A*02: 01-restricted NY-ESO-1 T-cell receptor (antigen-specific T cells). Antigen-specific T cells were preconditioned with belzutifan for 48 hours under 1% oxygen (hypoxia), after which RCC cells were stained with CellTrace Far Red and co-cultured with the T cells at an effector-to-target ratio (E: T) of 0: 1 or 1: 1 for 12 hours. Cytotoxic activity was quantified by flow cytometry using Annexin V and propidium iodide staining and gating on doublet-excluded, CellTrace-positive target cells. Specific lysis (%) was calculated as (1 − viability at an E: T = 1: 1 / viability at an E: T = 0: 1) × 100. Statistical significance was determined by two-sided paired t tests. Results: Treatment of 786-O-G323E and 786-O-WT cells with belzutifan for 12 hours in the absence of T cells did not reduce tumor-cell viability. Belzutifan-preconditioned T cells exhibited significantly higher specific lysis of both 786-O-G323E and 786-O-WT cells than DMSO-treated controls (p = 0. 0385 and p = 0. 0256, respectively). These results suggest belzutifan may enhance T-cell cytotoxicity. Conclusions: This study demonstrates that belzutifan exposure does not intrinsically impair antigen-specific T-cell cytotoxicity against RCC target cells in our hypoxic model antigen system. This finding supports the biological feasibility of combination therapy of belzutifan with ICIs in RCC. Citation Format: Soki Kashima, Katrina Madsen, Hanna Soulati, Zachary Yochum, Lena Wirth, Vivien Moritz, Marc Machaalani, Fed Ghali, Patrick Kenney, Adebowale Adeniran, Michael Hurwitz, Toni K. Choueiri, David A. Braun. Investigation of the impact of the HIF-2α inhibitor belzutifan on T-cell cytotoxicity in renal cell carcinoma (RCC) abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₂): Abstract nr A021.