Fig. 3: D4 CAR T cells with a shorter spacer domain significantly improve its reactivity against low GPC1-expressing tumor cells.
a Schematics of D4-IgG4 hinge(H)-based CAR with different length of spacers (CH3, 103 aa and CH2CH3, 216 aa). A shorter IgG4H (12 aa) was used to replace the original CD8H (45 aa). CD28 transmembrane (TM) domain was used in all D4-IgG4 hinge-based CARs. The illustration was created with BioRender.com. b Transduction efficiency of above D4 CAR constructs. Data are representative of two independent experiments. c The D4-IgG4H CAR T cells showed equally potent reactivity in high GPC1-expressing 2B9 cells, whereas demonstrated the best cytolytic activity when co-cultured with low GPC1-expressing T3M4 cells for 24 h. n = 3 independent experiments. ***p < 0.001, two-tailed unpaired Student’s t test. d Measurement of IFN-γ and IL-2 secretions in co-cultured supernatants with T3M4 cells at E:T ratio of 6:1. n = 3 independent experiments. *p = 0.016, **p < 0.01, ***p = 0.00017, two-tailed unpaired Student’s t test. e Experimental schematic (created with BioRender.com). T3M4 tumor-bearing NSG mice were treated with i.p. injection of 10 million mock T cells, D4-CD8H-CD8TM CAR T cells, D4-IgG4H-CD28TM CAR T cells, D4-IgG4H-CH3-CD28TM CAR T cells, and D4-IgG4H-CH2CH3-CD28TM CAR T cells on day 10 after tumor cell inoculation. n = 6 mice/group. f D4-IgG4H-CD28TM CAR T cells rapidly eliminated T3M4 tumor cells in mice, the ones with intermediate and long spacers only controlled tumor growth. g Tumor bioluminescence as photons per second in mice treated in (f). h Kaplan–Meier survival curve reveals a significant extended survival of mice receiving D4-IgG4H-CD28TM CAR T cells. ns, p = 0.2597, log-rank test. The CAR T cells used in this figure were produced using the donor 2’s PBMCs. Values represent mean ± SEM. Source data and exact p values are provided in the Source data file.
