Rab5 expression enables recycling of unbound CARs back to the CART surface. (a and b) Representative flow cytometry plots of CAR expression in CD4⁺ CARTs during CAE assay, detected by labeled CD19 protein. Data are representative of three independent experiments with samples from unique healthy donors. (c) Western blot showing CAR molecules in early endosome fraction purified from mCherry-fused 19BBz-tEGFR and 19BBz-Rab5 CARTs collected during the third-round CAE assay at the indicated time points. Data are representative of two independent experiments using samples from different healthy donors. (d and e) See Fig. S4 a for detailed workflow. Measurement of CD19-bound FLAG-CAR (FLAG⁺FMC63^-) and CD19-unbound FLAG-CAR (FLAG⁺FMC63⁺) on CARTs, indicated by representative flow cytometry plots (d) and summary data (e). Data are representative of three independent experiments with samples from different healthy donors. (f and g) CD19-unbound CARs were measured with FLAG-tag–fused control or Rab5-CARTs during the CAE assay. CARTs were gated on APC-FLAG⁺ populations before subsequent analysis of unbound CAR levels via anti-CD19 antibody staining using flow cytometry. Data are representative of two independent experiments with samples from unique healthy donors. (h) Surface CAR expression level measured staining of FLAG Ab after 48 h of the third-round CAE assay. Data are representative of three independent experiments. (i and j) Confocal imaging of the subcellular CAR (red) distribution 24 h after 19BBz-mCherry-tEGFR or -Rab5 CARTs co-cultured with K.19.GFP (green) (i), quantification of the percentage of mCherry-CAR internalization into tumor cells (j). Data are representative of three independent experiments with samples from unique healthy donors. (k and l) TEM images revealing the subcellular localization of engineered APEX2-tagged CAR molecules (black electron-dense deposits; k) within tumor cells. K.19.GFP cells were co-cultured with APEX2-fused control or Rab5 CARTs for 24 h, followed by T cell depletion and fixation. Samples were incubated with DAB and hydrogen peroxide to generate electron-dense APEX2 reaction products, and TEM images of K.19.GFP cells were captured. Quantification of the mean number of APEX2-CAR–positive vesicles per tumor cells is shown (l). Experiments are representative of five independent experiments. Error bars show mean ± SEM. Statistical comparisons were made using unpaired t tests. ***P < 0.001. Source data are available for this figure: SourceData F4.