Figure 5 from Nanobody-Engineered CLL-1 CAR T Cells: Optimizing Tumor-Specific Cytotoxicity and Minimizing Off-Tumor Toxicity

Nanobody-based CLL-1 and CD33 CAR T cells exhibit sustained cytotoxicity against antigen-expressing tumor cells. CLL-1 CAR T cells, CD33 CAR T cells, and untransduced (Unt.) T cells were cocultured with U937-mKate2 cells, an AML cell line with high CLL-1 and CD33 expression, at varying E:T ratios ranging from 10:1 to 0.33:1. Cytotoxicity was continuously monitored for 42 hours using the IncuCyte live-cell imaging system, with target cells labeled with mKate2 far-red fluorescent protein. To assess serial killing capacity, CAR T cells were rechallenged with fresh U937-mKate2 cells three consecutive times. CLL-1 and CD33 CAR T cells demonstrated comparable sustained killing efficiency even at low E:T ratios. Unt. T cells were included as a control to evaluate nonspecific killing. Shown are the mean values from four images acquired by the IncuCyte at each time point and condition, from one representative experiment out of three independent repeats with three different T-cell donors.