Panel a shows a schematic representation highlighting the scFv regions targeting CD19, mesothelin, and BCMA antigens. Panel b presents flow cytometry histograms showing intracellular Rab5 expression in control and Rab5-CARTs during a CAE assay. Panel c shows minimal gene expression changes at D9 baseline condition. Panel d shows CAE induces significant transcriptional shifts with upregulated immune-related genes. Panel e shows repeated CAE increases activation marker expression on T cells. Panel f shows CAE elevates exhaustion markers indicating progressive T cell dysfunction. Panel g contains flow cytometry plots illustrating CD19 trogocytosis at different time points for 19BBz-tEGFR and 19BBz-Rab5 CARTs.
Rab5 does not affect CART activation and short-term antigen capture. (a) Schematic representation of Rab protein expression 19BBz, MESOBBz, and BCMABBz CAR constructs. These constructs are the same with substituted scFv regions targeting CD19, mesothelin, and BCMA antigens, respectively. (b) Intracellular flow cytometry staining of Rab5 expression in both control and Rab5-CARTs during CAE assay. Data are representative of two independent experiments with samples from independent healthy donors. (c and d) Volcano plots of differential gene expression in control and Rab5 CARTs, from bulk RNA-seq. Significantly (P value <0.05) upregulated genes (>twofold), downregulated genes (≤twofold), and unchanged genes are highlighted in green, black, and grey, respectively. Data are representative of four samples from different donors. (e and f) Expression levels of the indicated surface markers (e) and the expression of T cell exhaustion markers (f) was comparable between 19BBz-tEGFR and -Rab5 CARTs. Data are representative of three independent experiments with samples from independent healthy donors. (g) Representative flow cytometry of CD3+CD19+ CARTs cultured with K.19.GFP cells. 19BBz-tEGFR or -Rab5 CARTs displayed comparable CD19 trogocytosis after short-term incubation at indicated time points. Data are representative of two independent experiments with samples from independent healthy donors.