GSE269885: RNA-seq analysis of human CD19 CAR T cells overexpressing RAB5

Full metadata and both raw and processed datafiles are available through NCBI GEO (accession #GSE269885)

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.

Human CD4+ and CD8+ T cells from four different healthy donors were used simultaneously for CART generation. Cells were transduced with either 19BBz-tEGFR or 19BBz-Rab5 vectors. To profile baseline differences between these two populations, CARTs expanded in vitro for 9 days were collected for analysis. To profile differences under repeated tumor challenges, beginning at day 9, CARTs were co-cultured with K.19.GFP cells at a 1:2 ratio (CARTs effector:target). Every 2 days, target cells were replenished to rechallenge CARTs. After the third round of target replenishment, at 48 hours post-plating, CD3+CAR+ cells were enriched. Dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec). T cells were isolated from target K562 cells with using the Dynabeads™ Untouched™ Human T Cells Kit (Invitrogen). Surface CAR-positive cells were sorted by flow cytometry. A portion of each sample was reserved to test Rab5 overexpression via immunoblotting. For RNA-sequencing analysis, while another portion with 1x106 cells from each sample were flash-frozen in liquid nitrogen and sent to BGI Genomics for sample QC, mRNA isolation, RNA library preparation and sequencing. To address potential variability in genes with low read count, filtering was performed to omit genes in which more than 4 samples had a read count <10. Genes with an adjusted p-value <0.05 were considered significant, and those with fold changes of >3 were defined as differentially expressed genes (DEGs) for the present study.