Panel a shows experimental timeline and CAR T cell infusion schedule overview. Panel b shows flow cytometry plots of CAR T cells before infusion. Panel c shows flow cytometry comparing 19BBz-tEGFR and 19BBz-Rab5 CAR T cells. Panel d shows increased CAR T cell frequencies after infusion in mice. Panel e shows quantification of CD4 positive and CD8 positive CAR T cells. Panel f shows representative flow plots highlighting CAR expression differences between groups. Panel g shows enhanced expansion of 19BBz-Rab5 CAR T cells observed. Panel h shows schematic of tumor model and experimental treatment workflow timeline. Panel i shows body weight changes in mice following tumor implantation. Panel j shows flow cytometry of tumor infiltrating CAR T cells populations. Panel k shows quantification of tumor burden and CAR T cell frequencies. Panel l shows flow cytometry of MESOBBz CAR T cells in tumors. Panel m shows CAR expression across CD8 positive and CD4 positive subsets. Panel n shows activation and exhaustion marker expression after CAR T infusion.
Rab5 expression augments the ability of CARTs to control solid tumors. (a) Experimental design of in vivo tumor challenge mouse model. 1 × 106 K.19.GFP cells mixed with Matrigel were s.c. injected to NSG mice on day 0. Tumors were imaged 6 days later, followed by tail i.v. injection with 1 × 106 19BBz CARTs on day 7. (b) Surface CAR expression level between 19BBz-tEGFR and -Rab5 CARTs before infusion. Data are representative of five independent experiments with samples from independent healthy donors. (c–e) Surface CAR expression and absolute CARTs number of CD4+ (c and e), and CD8+ CARTs (d and e) isolated from tumors after 14 days CARTs infusion. Data are representative of two independent experiments, n = 5–7 mice for each group. (f) Peripheral blood CART expansion was measured 14 days after infusion of 19BBz-tEGFR or -Rab5 CARTs into K.19.GFP tumor-bearing mice. Data are representative of two independent experiments, n = 5–7 mice for each group. (g) CAR expression in CARTs from samples in f 14 days after infusion. Data are representative of two independent experiments, n = 5–7 mice for each group. (h) Schematic of mesothelin tumor mouse model. 1 × 106 EMMESO-GFP or AsPC-1 cells were s.c. injected to NSG mice. Tumor size reached around 150 mm3 after 20 days, 0.5 × 106 NTD, MESOBBz-tEGFR or MESOBBz-Rab5 CARTs were infused i.v. on day 21. (i) Body weight of individual EMMESO-tumor–bearing mice was monitored weekly from the day of MESOBBz CART infusion. Data are representative of three independent experiments, n = 5–11 mice for each group. (j and k) Expansion of tumor-infiltrating MESOBBz CARTs was detected after 21 days infusion using anti-huCD45 and anti-huCD3 antibodies. Data are representative of two independent experiments, n = 5 mice for each group. (l and m) CAR surface expression in both CD4+ and CD8+ intratumoral CARTs after 21 days infusion was assessed by flow cytometry (l), and representative data for the percentage of intratumoral CARTs are shown (m). Data are representative of two independent experiments, n = 5 mice for each group. (n) Exhaustion markers on tumor infiltrating MESOBBz-tEGFR or -Rab5 CARTs were determined 21 days after infusion. Data are representative of two independent experiments, n = 5 mice for each group. Error bar show mean± SEM. Statistical comparisons were made using unpaired t tests. **P < 0.01, ***P < 0.001, and ****P < 0.0001.