Fig. 7.

The antitumor activity of low-affinity dual-targeted CAR-T cells. a Cytokine secretion was assayed in supernatants from co-cultures of low-affinity HER2 CAR-T cells with or without the PD-L1 CCR. Bar charts show data from three healthy donors, which are represented as the mean ± SEM, for IL-2 and IFN-γ (NS, no significance; ** P < 0.01; **** P < 0.0001, analyzed using a paired Student’s t-test). b Cytokine release was assayed in supernatants from CAR-CD4⁺ T cells with or without the PD-L1 CCR. Each value represents the mean ± SEM of triplicates for IL-2 and IFN-γ (NS, no significance; * P < 0.05; ** P < 0.01; *** P < 0.001; analyzed using Student’s t-test). c Specific killing of PD-L1^−/+ SKOV3 target cells by low-affinity HER2 CAR-T cells equipped with or without PD-L1 CCR was measured. The engineered T cells were co-cultured with PD-L1^+/− SKOV3 cells at the indicated E:T ratios after 18 h of incubation. Each value represents the mean ± SEM of technical triplicates (NS, no significance; * P < 0.05; ** P < 0.01; *** P < 0.001, analyzed using Student’s t-test). d-f The antitumor activity of low-affinity dual-targeted CAR-T cells in vivo. d Schematic diagram of T cells administration protocol. PD-L1^+/− SKOV3 cells were injected subcutaneously into the flank of mice on day 0. Ten days post-inoculation, the mice were randomized into four groups (n = 5 mice/group) and treated with 4 million control T cells, low-affinity HER2 CAR-T cells (CD4: CD8 = 1: 1), dual-targeted CAR-T cells (CD4: CD8 = 1: 1) or an equal volume of PBS. Tumor size was measured with calipers every 5 sssor 10 days. e Tumor growth curve for mice implanted with SKOV3 tumor xenografts. f Tumor size measurement for mice engrafted with PD-L1⁺SKOV3 tumor xenografts