Comparison of CD19/CD22/CD3 (CC) and blinatumomab in vitro and in vivo. Cytotoxicity comparison of CD19/CD22/CD3 (CC) and blinatumomab against Nalm6, Nalm6-KO19, and Nalm6-KO22 cells after 24 hours of incubation using expanded T cells as effector cells at an E:T ratio of 1:1 (A) or peripheral blood mononuclear cells (PBMCs) as effector cells at an E:T ratio of 10:1 (B). Experiments were performed in triplicate and repeated 3 times with similar results. (C) Schematic representation showing the experimental design. Nalm6-KO19 and Nalm6-KO22 at equal ratios were mixed immediately with 10 × 10⁶ fresh PBMCs from healthy donor #5 before IV injection into NCG mice (n = 5/group). Two hours later, intraperitoneal treatments with blinatumomab or CD19/CD22/CD3 (CC) were initiated at 5.33 nmol/kg and conducted daily for 8 consecutive days. On day 5, an additional injection of fresh PBMCs (10 × 10⁶) was added to supplement the effector cells. Similar results were obtained in 2 independent experiments. (D) Representative bioluminescence images of mice treated with blinatumomab or CD19/CD22/CD3 (CC). Colors indicate the intensity of luminescence (red, highest; blue, lowest). (E) Average radiance quantification (p/s/cm²/sr) of the luminescence is shown. Statistical significance was calculated using the Dunnett multiple comparisons test. (F) Survival curves of mice treated with blinatumomab or CD19/CD22/CD3 (CC). The log-rank (Mantel‒Cox) test was used to calculate significance. ∗∗P < .01. Quantitative analysis of leukemia burden (G) and T-cell persistence (H) in the peripheral blood of mice was performed on day 16 and day 20. Nalm6-KO22 and Nalm6-KO19 cells were defined as GFP⁺CD19⁺ and GFP⁺CD22⁺, respectively. Data are plotted as the mean ± SD. Statistical significance was calculated using the Tukey multiple comparisons test. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001; ns, not statistically significant (≥.05).