Figure 4.

Multitargeting with AdCAR T cells prevents immune evasion in lymphoma. (a) Non-transduced activated T cells with or without 1 ng/mL LLE-CD19 mAb, AdCAR T cells with or without 1 ng/mL LLE-CD19 mAb +/− 1 ng/mL LLE-CD20 mAb, or conventional CD19CAR T cells were incubated with JeKo-1 (left), Raji (middle), and Daudi (right) at the indicated E:T ratios. Target cells only were incubated with LLE-CD19 mAb at 1 ng/mL. Target cell lysis was determined by LCA after 48 h. A statistical analysis is presented for the comparison of the AdCAR T cells + LLE-CD19 mAb versus the activated T cells. (b) AdCAR T cells with 1 ng/mL LLE-CD19 mAb or 1 ng/mL LLE-CD19 mAb + 1 ng/mL LLE-CD20 mAb, or conventional CD19CAR T cells were incubated for 48 h with a CD19 knockout variant of JeKo-1 (JeKo-1_CD19KO) at the indicated E:T ratios. Target cell lysis was determined by LCA after 48 h. A statistical analysis is presented for a comparison of the combinatorial targeting (CD19 + CD20) versus the monotargeting (CD19). (c,d) To demonstrate the flexibility of the AdCAR system, AdCAR T cells were incubated with either JeKo-1 (c) or Raji (d) at an E:T ratio of 1.25:1 with 1 ng/mL of the indicated LLE-mAbs. Alternative target antigens can be utilized by AdCAR T cells. Combinatorial targeting was feasible and increased the engagement of the AdCAR T cells with the cancer cells. Target cell lysis was determined by LCA after 48 h. The statistical analysis demonstrates the superiority of CD19 as a target antigen compared with the alternative antigens CD20 and CD79B in JeKo-1. Targeting CD19 mediated the strongest cytolytic effect in Raji. Data shown in (a,b) represent the mean ± SEM of (n = 3) independent experiments in triplicate from three different donors. Data shown in (c,d) represent the mean ± SEM of (n = 3) independent experiments from three different donors. Statistical analysis was performed using one-way ANOVA and Tukey’s post-hoc test. ns, not significant. *, p ≤ 0.05. **, p ≤ 0.01. ***, p ≤ 0.001. ****, p ≤ 0.0001.