Fig. 5

In vivo cytotoxic effect of sdCAR-T cells toward the cognate tumor cells. a Schematic of the mouse treatment strategy used in the in vivo experiment. Matched MSLN⁺/CEA⁺ K562 cells described previously were injected into nude mice intraperitoneally (i.p.). Effector cells, switch molecules, or PBS were injected i.p. at the indicated time point. After 48 h, both target cells were recovered from peritoneal lavage and quantified by flow cytometry. b Various concentrations of FHBM were added to mice. The degree of cytotoxicity of sdCAR-T cells together with saturated FHBM (0.5 mg/kg) matched the level observed with the MζBB CAR-T cells that triggered significant cytotoxicity for cognate tumor cells. c At the end of the experiment, we quantitatively analyzed the remaining cognate and non-cognate tumor cells by flow cytometry, respectively. sdCAR-T cells eliminated the cognate tumor cells in mice treated with FHBM, comparable to the cytotoxicity of MζBB CAR-T cells. d Ratios of surviving MSLN⁺ K562:CEA⁺ K562 in each sample. Cognate target cells were killed by sdCAR-T cells only in the presence of FHBM. We observed similar results for MζBB CAR-T cells. (n = 5, error bars denote standard deviation.) e–h The sdCAR-T cells produced lower levels of cytokines. During the experiment, sdCAR-T cells released IL-2 (~ 1300 pg/mL), IFNγ (~ 30 ng/mL), IL-6 (~ 1160 ng/mL), and TNFα (~ 380 pg/mL) only in the presence of FHBM. MζBB CAR-T cells always released high levels of cytokines, including ~ 2600 pg/mL of IL-2, ~ 60 ng/mL of IFNγ, ~ 2216 ng/mL of IL-6, and ~ 647 pg/mL of TNFα. (n = 5, error bars denote standard deviation)