Panel a shows flow cytometry plots of CAE assay over time. Panel b shows line graph of tumor burden progression in mice. Panel c shows tumor growth curves comparing different CAR T treatments. Panel d shows excised tumors illustrating reduced size with Rab5 treatment. Panel e shows flow cytometry plots measuring tumor cell populations. Panel f shows quantification of GFP positive tumor cells after treatments. Panel g shows flow cytometry plots of CAR T cell infiltration. Panel h shows quantification of CD3 positive CAR T cells infiltration. Panel i shows tumor growth curves across extended experimental timeline. Panel j shows excised tumors confirming reduced tumor burden in Rab5. Panel k shows flow cytometry plots of CAR expression on T cells. Panel l shows quantification of CAR positive cells across treatment groups. Panel m shows flow cytometry plots of CD4 positive CAR T cells. Panel n shows quantification of CD4 positive CAR T cells frequencies.
Rab5 expression enhances the ability of CARTs to control solid tumors. (a and b) Primary human T cells were activated and transduced with MesoBBz-CAR expressing either tEGFR or Rab5. After 18 days of culture, 1 × 106 CAR+ T cells were co-cultured with 2 × 106 GFP+ EMMESO cells. Representative flow cytometry plots (a) and summary (b) of GFP+ EMMESO populations throughout the CAE assay. Data are representative of three independent experiments with samples from different healthy donors. (c) Individual EMMESO tumor sizes in mice infused with the indicated MESOBBz CARTs, measured weekly. Data are representative of three independent experiments, n = 5–11 mice for each group. (d) Representative images of EMMESO tumor tissues isolated 21 days after infusion from mice treated with MESOBBz-tEGFR or MESOBBz-Rab5 CARTs. Data are representative of two independent experiments, n = 5 mice for each group. (e and f) Tumor-bound MESOBBz CARs, detected in tumor-infiltrating control or Rab5 CARTs 21 days after infusion, using tumor samples from d. Data are representative of two independent experiments, n = 5 mice for each group. (g and h) Surface CAR expression 21 days after MESOBBz-tEGFR or MESOBBz-Rab5 CARTs infusion, assessed by anti-G4S linker antibody staining in tumor samples from d. Data are representative of two independent experiments, n = 5 mice for each group. (i) Individual AsPC-1 tumor sizes in mice infused with the MESOBBz CARTs, measured weekly. NSG mice were injected s.c, with 1 × 106 AsPC-1 cells. After 21 days, tumor-bearing mice were infused with 0.5 × 106 MESOBBz-tEGFR or MESOBBz-Rab5 CAR T cells. Data are representative of two independent experiments, n = 10 mice per group. (j) Representative images of AsPC-1 tumor tissues isolated 21 days after MESOBBz-tEGFR or MESOBBz-Rab5 CARTs infusion. Data are representative of two independent experiments, n = 5 mice for each group. (k and l) Expansion of tumor-infiltrating CARTs was assessed 21 days after infusion using tumor samples from j. (m and n) Surface CAR expression on tumor-infiltrating CARTs was measured 21 days after infusion using tumor samples from j. Data are representative of two independent experiments, n = 5 mice for each group. Error bars show mean ± SEM. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.