Sequential therapy with tafasitamab and CART19 cell decreased the severity of CRS and increased the antitumor effect of CART19. (A) Experimental scheme. NSG mice were first treated with 30 mg/kg of busulfan via IP injection. After 24 hours, the mice were injected with 5 × 10⁶ of leukemic blasts derived from patients with R/R ALL. The mice were then monitored for tumor burden via PB sampling. Once human CD45⁺ cells within mouse blood reached >10 cells per μL, the mice were randomized according to the burden of human CD45⁺ cells to receive (1) IgG control or (2) 5 mg tafasitamab on day −7. Tafasitamab and IgG controls were administered via IP injection. On day 0, IgG control and tafasitamab groups received 3.5 × 10⁶ of luciferase⁺ CART19. The expansion of CART19 was monitored via serial BLI, and CRS was monitored through the weight and well-being of the mice. (B) Mice were bled before CART19 cell infusion, and the tumor burden was reassessed. The leukemic blasts were determined by human CD45⁺, mouse CD45⁻, and human CD20⁺ cells (∗P < .05, t test: n = 3-6 per group). (C) CD19 expression in leukemic blasts was assessed using flow cytometry. CD19 absolute counts were determined using Quantum Simply Cellular kits (∗P < .05, t test: n = 3-5 per group). (D) The percentage reduction in the weights from baseline is shown. The first and second asterisks or “n.s.” are showing the statistical comparisons between IgG control vs 5 mg/kg tafasitamab at day −7 or IgG control vs untreated xenografts, respectively ∗∗P < .01, ∗∗∗∗P < .0001, 2-way ANOVA). (E) Analysis of CART19 cell expansion in vivo. IgG control and tafasitamab groups were imaged with bioluminescence on days 1 and 2 (∗P < .05, ∗∗P < .01, t test). (F) On day 4 of CART19 treatment, mice were bled and cytokines were analyzed with multiplex (∗∗∗P < .001, ∗∗∗∗P < .0001, t test). (G) Kaplan-Meier curve is shown. IgG control vs 5 mg/kg tafasitamab at day −7 HR, 17.81; 95% CI, 3.805 to 83.40; ∗∗P = .0003 (log-rank test), 5 mg/kg tafasitamab vs untreated xenografts, 0.04569; 95% CI, 0.008027 to 0.2601; ∗∗∗P = .0005 (log-rank test), IgG control vs untreated xenografts HR, 13.64; 95% CI, 2.855 to 65.17, ∗∗P = .0011 (log-rank test). (H) The weights of spleens are shown. At the end of the experiments, the mice were euthanized and the spleens were harvested (∗P < .05, 1-way ANOVA). (I) The sizes of spleens are shown. Splenic cells were analyzed using flow cytometry. Leukemic blasts and CART19 were defined as human CD45⁺, mouse CD45⁻, human CD20⁺, and human CD45⁺, mouse CD45⁻, human CD3⁺, respectively.