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RNA-seq analysis of human CD19 CAR T cells overexpressing RAB5

ABSTRACT: Chimeric antigen receptor (CAR) T cells (CARTs) are engineered T cells designed to target specific molecules on the surface of cancer cells. Using a continuous antigen exposure (CAE) assay to model this interaction, we observed that tumors can capture CAR molecules, leaving the CARTs devoid of surface-expressed CARs and unable to kill tumors after repeated challenges. Overexpression of Rab5, a key regulator of endocytosis, prevents the loss of CARs and enhances CARTs activity. Rab5 significantly increased the endocytic recycling of T cells without causing other perturbations to CART function. Interestingly, we observed membrane protrusions on the CART cell surface, which disappeared after multiple tumor challenges. Rab5 overexpression maintained these protrusions after multiple tumor engagements, and the presence of membrane protrusions correlated with tumor clearance. In vivo, Rab5-expressing CARTs demonstrated improved activity and were able to clear an otherwise recalcitrant mesothelin-expressing solid cancer. These findings suggest that engineering CARTs with Rab5 overexpression could enhance the clinical efficacy of CAR T cell therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE269885 | GEO | 2026/04/10

REPOSITORIES: GEO

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Project description:BACKGROUND: Chimeric antigen receptor (CAR) T cells have achieved remarkable clinical success. However, up to 50% of CAR T cell-treated leukemia patients relapse and long-term survivor data indicate that CART cell persistence is key to enforcing relapse-free survival. Unfortunately, ex vivo expansion protocols often drive metabolic and functional exhaustion, reducing in vivo efficacy. Preclinical models have demonstrated that redirecting metabolism can improve in vivo T cell function. Here, we hypothesized that exposure to an agonist targeting AMP-activated protein kinase (AMPK) would create CARTs capable of increased in vivo function and enhanced leukemia clearance. METHODS: CART cells were generated from healthy human donor T cells via lentiviral transduction, followed by exposure to either Compound 991 or DMSO for 96 hours, followed by a 48-hour washout. During and after agonist treatment, T cells were harvested for metabolic and functional assessments. To test in vivo efficacy, immunodeficient mice were injected with luciferase+ NALM6 leukemia cells, and one week later with 991- or DMSO-expanded CARTs. Leukemia burden and anti-leukemia efficacy was assessed via radiance imaging and overall survival. RESULTS: Compound 991 treatment activated AMPK without limiting cellular expansion, and increased both mitochondrial density and handling of reactive oxygen species (ROS). Mechanistically, 991 treatment mimicked nutrient starvation, with increased autophagic flux and generation of mitochondrially-protective metabolites in treated cells. Importantly, receipt of 991-exposed CARTs significantly improved in vivo leukemia clearance and prolonged recipient survival, likely as a result of elevated activation and increased CD4+ T cell yields at early times post-injection. DISCUSSION: Ex vivo expansion is necessary to generate sufficient cell numbers, but sustained activation and differentiation often negatively impact in vivo persistence and function. Here, we demonstrate that promoting AMPK activity during in vitro CART expansion metabolically reprograms cells without limiting T cell yield, increases early activation marker expression following in vivo transfer, and ultimately enhances anti-leukemia efficacy. Importantly, Compound 991 treatment achieves these results without further modifying the expansion media, changing the CART construct, or genetically altering the cells. Altogether, these data highlight AMPK agonism as a potent and readily translatable approach to improve the metabolic profile and in vivo efficacy of adoptively transferred T cells.

Project description:Chimeric antigen receptor (CAR) T cell therapy have demonstrated remarkable success in treating B-cell malignancies that are refractory to standard therapies; however, preclinical and clinical studies have demonstrated that CAR T cell efficacy is greatly reduced against antigen-low tumors. This was exemplified in a clinical trial of CD22-directed CAR T cells where post-CAR relapses were driven by a decrease in the surface expression of the CD22 antigen. To address the poor response of CAR T cells to antigen-low tumor cells, we performed global phosphoproteomics using a SILAC/mass spectrometry approach to examine signal transduction events within CAR T cells in response to high- and low-levels of CD22 antigen. Stimulation with low levels of CD22 antigen resulted in decreased activation of several canonical T cell signaling pathways in CAR T cells, suggesting poor utilization of LAT. To overcome this inefficiency of LAT activation, we designed a bicistronic construct consisting of a clinically active 2nd generation CD22-BBz CAR along with a novel Adjunctive LAT Activating CAR incorporating the intracellular signaling domain of LAT (ALA-CART). ALA-CART cells demonstrated enhanced phosphorylation of LAT and restored downstream signaling in response to low levels of CD22. In xenograft models, ALA-CART cells completely eradicated CD22-low leukemia, significantly extending survival of mice in comparison to the clinically-active, standard CD22-BBz CAR T cells. Compared to the standard CD22-BBz CAR T cells, ALA-CART cells exhibited transcriptional differences in the resting state reflecting a less differentiated state, which corresponded to an enrichment of T stem cell memory cells and enhanced in vivo persistence. Thus, through the identification of CAR signaling deficits, we rationally designed the ALA-CART platform which improves the sensitivity and persistence of CAR T cells to target antigen-low cells, overcoming a mechanism of resistance to standard CAR therapies.

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RNA-seq analysis of human CD19 CAR T cells overexpressing RAB5

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