Figure 7.

The B2ARM CAR with exhibits superior efficacy in vivo. (A) NSG mice were intradermally injected on the abdomen with 8 x 10⁶ RPMI-8226 cells with overexpressed TGF-β (n = 10 in all groups except untreated, n = 5). On day 17 after tumor injection, 5 x 10⁶ CAR+ T-cells were intravenously injected. The differences in CAR expression levels were normalized by adjusting the total number of infused T-cells. On day 7 after T-cell infusion, 5 mice from each group (except the untreated group) were sacrificed for tumor harvest, while the rest were monitored for (B) tumor progression, (C) survival, and (D) weight change. (E) the percentage of CD3+CAR+ T cells in the tumor homogenates was determined by flow cytometry following tumor harvest. Tumor homogenates were incubated at 37°C for 5 hours in the presence of brefeldin A, and (F) IFN-γ, (G) TNF-α, and (H) IL-2 cytokine production in CD3+ cells was determined by intracellular staining and flow cytometry analysis. Statistical significance was determined by one way ANOVA with Tukey’s post-hoc test, *p<0.05, **p<0.01, ****p<0.0001. The percentage of Ki67, (I), Granzyme B, (J), and PD-1, (K) expression in CD3+ cells from TIL populations was determined by intracellular or surface staining and flow cytometry in tumor homogenates following tumor harvest. Statistical significance was determined by Student t-test, *p<0.05, **p<0.01, ns, non-specific.