Figure 6 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 effectively eliminate human AML in vivo in a xenograft model. A, Schematic representation of the U937 xenograft model. To establish leukemia in vivo, NSG mice were intravenously injected via the tail vein with 1 × 106 U937-mKate2-Luc AML cells expressing firefly luciferase on day 0. Disease engraftment was confirmed on day 7 using BLI, and mice were subsequently randomized into four treatment groups. On day 7, mice received i.v. injections of either CLL-1 CAR T cells (1 × 107), CD33 CAR T cells (1 × 107), untransduced (Unt.) T cells (1 × 107), or PBS as a control. AML burden was longitudinally monitored via serial BLI, with bioluminescence radiance serving as a surrogate marker for tumor progression. B, Tumor burden assessment through BLI. Representative BLI images of mice from each treatment group were taken at key time points: before treatment (day 7), early response (day 14), and throughout the study period until day 63 after tumor injection. Changes in tumor burden were visualized and quantified to assess therapeutic efficacy. C, Kaplan–Meier survival analysis. Survival curves were generated to compare overall survival between treatment groups. Statistical analyses were performed using the log-rank (Mantel–Cox) test to determine significant differences in survival outcomes. D, Quantitative analysis of bioluminescence signal over time. Serial BLI measurements were used to track leukemia progression and response to therapy. The bioluminescent signal was quantified and plotted for each treatment group, represented as mean radiance (photons/second/cm2/sr) ± standard deviation (SD). E, Body weight monitoring was initiated on day 14, 1 week following CAR T-cell administration, as a measure of overall treatment tolerance and potential toxicity. Mouse body weight was recorded throughout the study to assess potential treatment-related toxicity. No significant weight loss was observed, indicating that CLL-1 and CD33 CAR T-cell therapy was well tolerated. F, The graph showed the CD8+ CAR expression in mouse blood at day 45. Tumor burden data used two-way ANOVA to determine the statistical significance between groups based on two or more independent variables. A t test was performed for CD8+ CAR detection to determine statistical significance. Each treatment group consisted of n = 5 mice. *, P < 0.0197; ****, P < 0.0001.