Fig. 7. Dimerization of D4 CAR leads to the enhanced T-cell function.

a Mutations of two cysteine residues in the hinge of D4-IgG4H-CD28TM CAR. b D4-IgG4H(mut)-CD28TM CAR T cells showed reduced binding to GPC1 though still had decent transduction efficiency determined by hEGFRt expression. Data are representative of two independent experiments. c D4-IgG4H-CD28TM CAR T cells lost the enhanced reactivity when both cysteine residues are mutated. n = 3 independent experiments. *p = 0.019, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test. d Representative western blot of CAR expression under non-reducing condition. Membranes were stained with CD3ζ antibody. Experiments were repeated with similar results. e Modeling of D4-CD8H-CD8TM and D4-IgG4H-CD28TM CARs. f Western blot analysis of T-cell signaling molecules of various D4 CAR T cells upon stimulation with T3M4 cells in the cytoplasm. g Nuclear localization of transcription factors upon stimulate with T3M4 cells in the nucleus. h Blots in (f) and (g) were representative of three experiments using T cells prepared from three different donors. The compiled data were quantified using ImageJ and normalized with mock control. *p = 0.041, one-tailed unpaired Student’s t test. i Proposed mechanism of action (created with BioRender.com). D4-IgG4H-CD28TM CAR T cells are dimerized to enhance the phosphorylation of PLCγ in the cytoplasm and subsequently promote canonical and non-canonical NF-κB signaling, leading to the promising antitumor efficacy in low GPC1-expressing solid tumors. The CAR T cells used in this figure were produced using the donor 2’s and donor 4’s PBMCs. Values represent mean ± SEM. Source data and exact p values are provided in the Source data file.