Figure 6.
CD19/CD22/CD3 (CC) can efficiently eliminate primary B-ALL patient tumor cells in vitro and in vivo. (A) Quantitative analysis of CD19 and CD22 antigen expression levels in 5 primary B-ALL patient samples by flow cytometry. (B) Cytotoxicity comparison of CD19/CD22/CD3 (CC) and bsAbs against 5 primary B-ALL cells after 24 hours of incubation with expanded T cells at an E:T ratio of 1:1. Experiments were performed in triplicate and repeated 3 times with similar results. (C) Inflammatory cytokines (IL-2, IFN-γ, and TNF-α) released from T cells cocultured with primary B-ALL cells in the presence of bsAbs or tsAb (100 pM) for 24 hours at an E:T ratio of 1:1 in triplicate. ∗P < .01, ∗∗P < .05, ∗∗∗P < .001. Values indicate the mean ± SD from the data of 3 independent experiments. (D) Scheme showing the experimental design. NCG mice (n = 5/group) were IV transplanted with 5 × 106 primary B-ALL cells from B-ALL Pt #6. Upon B-ALL engraftment detectable in peripheral blood (day 20 for B-ALL Pt #6), mice were randomized, and 2 doses of expanded T cells were IV injected at the indicated time points on day 2 and day 7, respectively. Human B-ALL cells and human T cells in peripheral blood were analyzed by flow cytometry and defined as mCD45-hCD45+hCD10+hCD3- and mCD45-hCD45+hCD10-hCD3+, respectively. Mice were intraperitoneally treated with bsAbs, bsAbs in combination, or tsAb (5.33 nmol/kg) daily for 7 consecutive days. Similar results were obtained in 2 independent experiments. Leukemic burden (E) and human T-cell persistence (F) in peripheral blood were measured over a 10-week follow-up period. (G) Body weight was monitored during treatment. (H) Survival curves of mice treated with bsAbs or tsAb. The log-rank (Mantel‒Cox) test was used to calculate significance. ∗P < .05. (I) TH1 (IL-2, IFN-γ, TNF-α) and TH2 (IL-4, IL-6, IL-10) cytokines released into the serum were evaluated 2 hours after the first drug infusion using a BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit. ∗P < .01, ∗∗P < .05, ∗∗∗P < .001; ns, not statistically significant (≥.05).

CD19/CD22/CD3 (CC) can efficiently eliminate primary B-ALL patient tumor cells in vitro and in vivo. (A) Quantitative analysis of CD19 and CD22 antigen expression levels in 5 primary B-ALL patient samples by flow cytometry. (B) Cytotoxicity comparison of CD19/CD22/CD3 (CC) and bsAbs against 5 primary B-ALL cells after 24 hours of incubation with expanded T cells at an E:T ratio of 1:1. Experiments were performed in triplicate and repeated 3 times with similar results. (C) Inflammatory cytokines (IL-2, IFN-γ, and TNF-α) released from T cells cocultured with primary B-ALL cells in the presence of bsAbs or tsAb (100 pM) for 24 hours at an E:T ratio of 1:1 in triplicate. ∗P < .01, ∗∗P < .05, ∗∗∗P < .001. Values indicate the mean ± SD from the data of 3 independent experiments. (D) Scheme showing the experimental design. NCG mice (n = 5/group) were IV transplanted with 5 × 106 primary B-ALL cells from B-ALL Pt #6. Upon B-ALL engraftment detectable in peripheral blood (day 20 for B-ALL Pt #6), mice were randomized, and 2 doses of expanded T cells were IV injected at the indicated time points on day 2 and day 7, respectively. Human B-ALL cells and human T cells in peripheral blood were analyzed by flow cytometry and defined as mCD45-hCD45+hCD10+hCD3- and mCD45-hCD45+hCD10-hCD3+, respectively. Mice were intraperitoneally treated with bsAbs, bsAbs in combination, or tsAb (5.33 nmol/kg) daily for 7 consecutive days. Similar results were obtained in 2 independent experiments. Leukemic burden (E) and human T-cell persistence (F) in peripheral blood were measured over a 10-week follow-up period. (G) Body weight was monitored during treatment. (H) Survival curves of mice treated with bsAbs or tsAb. The log-rank (Mantel‒Cox) test was used to calculate significance. ∗P < .05. (I) TH1 (IL-2, IFN-γ, TNF-α) and TH2 (IL-4, IL-6, IL-10) cytokines released into the serum were evaluated 2 hours after the first drug infusion using a BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit. ∗P < .01, ∗∗P < .05, ∗∗∗P < .001; ns, not statistically significant (≥.05).

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