Figure 3.
PRDM1-ablated CD19-targeting CAR-T cells show superior persistence and antitumor activity in vivo. (A-C) CD19-targeting CAR-T cells electroporated with or without Cas9 and sgRNAs against PRDM1 were infused into tumor-free NSG mice. The frequency of human T cells in the PB was monitored at the indicated time points (B) (unpaired 2-tailed Student t test). Kaplan-Meier analysis for overall survival after CAR-T cell infusion (C) (n = 7 mice for each group, log-rank test). The data are a composite of 2 independent experiments. (D) The PRDM1 knockout efficiency in infusion products and the persisting CAR-T cells within the spleen was analyzed (n = 7, paired 2-tailed Student t-test). (E) Cytolytic activity of control or PRDM1-knockout CAR-T cells against the indicated target cells was analyzed with flow cytometry (n = 3 cultures, unpaired 2-tailed Student t test for each condition). Representative data of 2 experiments. (F-G) Cytolytic activity of control or PRDM1-knockout CAR-T cells against NALM6 (F) or K562-CD19 (G) at the indicated time points (n = 3-4 cultures, one-way ANOVA with multiple comparisons test). (H-L) NSG mice were intravenously infused with NALM6-GL, and then with CAR-T cells with or without PRDM1 knockout 10 days later (n = 10 mice for each CAR). (I) The frequency of human T cells in the PB was analyzed at the indicated time points (unpaired 2-tailed Student t test of the log-transformed values for each time point). (J) Total flux in the whole body was measured by in vivo bioluminescence imaging (unpaired 2-tailed Student t test of the log-transformed values). (K) Kaplan-Meier analysis for leukemia-related survival after NALM6-GL infusion (log-rank test). In (H-K), representative data of 2 independent experiments are shown. (L) The PRDM1-knockout frequency was compared between infusion products and T cells within the spleen of the mice that developed xenogeneic GVHD (n = 4, paired 2-tailed Student t test). (M-N) NALM6-GL-engrafted NSG mice were transplanted with control or PRDM1-knockout CAR-T cells generated in the same protocol as that shown in (H). The frequency of CD8+ or CD4+ CAR-T cells (M) (n = 6, unpaired 2-tailed Student t test of the log-transformed values) or those with TSCM and TCM phenotypes (N) (n = 6, unpaired 2-tailed Student t test for each time point) in the PB was determined by flow cytometry. (O-R) NSG mice subcutaneously inoculated with A375-CD19 (day 0) were treated with CAR-T cells with or without PRDM1 knockout (day 7). The mice were rechallenged with A375-CD19 (day 24) and monitored for tumor progression (n = 9 mice for each group). The data shown are serial monitoring of tumor volume (P), relapse-free survival (Q) (log-rank test), and the frequency of human T cells at the indicated time points (R) (unpaired 2-tailed Student t test of the log-transformed values). In B, E, F, G, I, J, M, N, and R, horizontal lines denote the mean values. NS, not significant.

PRDM1-ablated CD19-targeting CAR-T cells show superior persistence and antitumor activity in vivo. (A-C) CD19-targeting CAR-T cells electroporated with or without Cas9 and sgRNAs against PRDM1 were infused into tumor-free NSG mice. The frequency of human T cells in the PB was monitored at the indicated time points (B) (unpaired 2-tailed Student t test). Kaplan-Meier analysis for overall survival after CAR-T cell infusion (C) (n = 7 mice for each group, log-rank test). The data are a composite of 2 independent experiments. (D) The PRDM1 knockout efficiency in infusion products and the persisting CAR-T cells within the spleen was analyzed (n = 7, paired 2-tailed Student t-test). (E) Cytolytic activity of control or PRDM1-knockout CAR-T cells against the indicated target cells was analyzed with flow cytometry (n = 3 cultures, unpaired 2-tailed Student t test for each condition). Representative data of 2 experiments. (F-G) Cytolytic activity of control or PRDM1-knockout CAR-T cells against NALM6 (F) or K562-CD19 (G) at the indicated time points (n = 3-4 cultures, one-way ANOVA with multiple comparisons test). (H-L) NSG mice were intravenously infused with NALM6-GL, and then with CAR-T cells with or without PRDM1 knockout 10 days later (n = 10 mice for each CAR). (I) The frequency of human T cells in the PB was analyzed at the indicated time points (unpaired 2-tailed Student t test of the log-transformed values for each time point). (J) Total flux in the whole body was measured by in vivo bioluminescence imaging (unpaired 2-tailed Student t test of the log-transformed values). (K) Kaplan-Meier analysis for leukemia-related survival after NALM6-GL infusion (log-rank test). In (H-K), representative data of 2 independent experiments are shown. (L) The PRDM1-knockout frequency was compared between infusion products and T cells within the spleen of the mice that developed xenogeneic GVHD (n = 4, paired 2-tailed Student t test). (M-N) NALM6-GL-engrafted NSG mice were transplanted with control or PRDM1-knockout CAR-T cells generated in the same protocol as that shown in (H). The frequency of CD8+ or CD4+ CAR-T cells (M) (n = 6, unpaired 2-tailed Student t test of the log-transformed values) or those with TSCM and TCM phenotypes (N) (n = 6, unpaired 2-tailed Student t test for each time point) in the PB was determined by flow cytometry. (O-R) NSG mice subcutaneously inoculated with A375-CD19 (day 0) were treated with CAR-T cells with or without PRDM1 knockout (day 7). The mice were rechallenged with A375-CD19 (day 24) and monitored for tumor progression (n = 9 mice for each group). The data shown are serial monitoring of tumor volume (P), relapse-free survival (Q) (log-rank test), and the frequency of human T cells at the indicated time points (R) (unpaired 2-tailed Student t test of the log-transformed values). In B, E, F, G, I, J, M, N, and R, horizontal lines denote the mean values. NS, not significant.

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