Abstract 4803: Adaptation of headless CAR T cells to tumor microenvironment induces enhance proliferation and anti-tumor activity against solid cancers

Cell therapy in solid tumors faces significant challenges due to the suppressive tumor microenvironment (TME), characterized by hypoxia and high interstitial pressure. These conditions affect gene and protein expression, contributing to tumor metastasis and drug resistance. Hypoxia dampens the anti-tumor T cell response by altering immune cell activation and function by modulating metabolic activity and the presence of immune suppressive cytokines, growth factors, and immune checkpoint molecules. The success of chimeric antigen receptor (CAR) T cell therapy in treating several hematopoietic malignancies has been challenging to replicate in solid tumors, in part because of T cell exhaustion and eventual T cell dysfunction. T cell reprogramming to adapt to TME is likely to represent a novel and essential aspect of enhancing anti-tumor activity in the TME. We used the Avatar and Avatar AI incubators (Xcell Bio) to precisely control oxygen concentration and pressure to simulate TME conditions to reprogram the headless CAR T cells (hCART). Bispecific antibody armed (ahCART) or unarmed (hCARTs) headless CAR T cells were acclimated under normal environment (NE) or TME conditions before coculturing with target cells. The effect of NE or TME conditions was determined on T cell proliferation, phenotypic changes, and functional activity of hCARTs. Functional readouts were measured by real-time cytotoxicity assay and cytokine profiles under NE or TME-like conditions (1-5% O2 + 2-5 PSI pressure). The phenotypic changes of ATC and hCARTs showed no changes in co-stimulatory receptor expression (41BB, CD40L, GITR, HVEM, ICOS, NFATc1, and TBET) on CD4 or CD8 T cells and co-inhibitory receptors (LAG2, LAIR, PD1, TIGIT, CTLA-4, FOXP3, GATA3 and BTLA) on CD8 T cells, except for reduced expression of certain co-inhibitory receptors on CD4 T cells under TME condition compared to NE condition. Acclimation of hCARTs under TME showed a delayed, but 100% cytotoxicity prolonged up to 7 days at low effector to target ratios of 1:1 or 2:1; non-acclimated hCART showed 100% cytotoxicity initially but declined in long-term or serial killing assays. There was no effect of TME preconditioning on cytokine profile (IL-2, GM-CSF, IFN-γ, TNF-α, and IL-6) compared to NE on hCART. These data suggest that TME reprogramming of hCARTs could be a promising approach to improve its effectiveness, especially in solid tumors. TME acclimation has the clinical potential to enhance the anti-tumor activity of cellular therapeutics. Citation Format: Archana Thakur, Amy Schienschang, Sarah Whitaker Amrhein, Yelena Bronevetsky, Shannon Eaker, Lawrence G. Lum. Adaptation of headless CAR T cells to tumor microenvironment induces enhance proliferation and anti-tumor activity against solid cancers [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 4803.