Figure 1.
Integration of a truncated CD19-specific CAR into CD3ζ, but not TRAC, conveys cytotoxicity in conventional T cells toward CD19+ leukemia cells. (A) full-length second-generation CAR protein (left) and virus-free knock-in strategies to integrate a full-length CAR into TRAC or a truncated CAR (truncCAR) into TRAC or CD3ζ. (B) Flow cytometry dot plots after knock-in. Transgene integration into TRAC or CD3ζ disrupts expression of the TCR/CD3 complex. (C) Relative cytotoxicity in coculture with (CD19+) Nalm-6 target cells and CD19 KO Nalm-6 control cells (VITAL assay). Calculation of relative cytotoxicity according to formula stated in methods section. (n = 2 biological replicates each in 2 technical replicates; ordinary one-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance). (D-G) Functional testing of CD3ζ truncCAR, T cells in comparison to TRAC CAR and LV CAR-T cells. (D) Mean fluorescence intensity (MFI) determined by flow cytometry as a measure of cellular CAR expression and normalized to each donor’s mean CAR MFI in the TRAC condition. (n = 7 biological replicates each in 2-5 technical replicates; mixed-effects analysis with Geisser-Greenhouse correction + Holm-Šídák multiple comparison test with individual variances computed for each comparison). (E) Relative cytotoxicity towards CD19+ cells assessed in a 6-hour VITAL assay. (mock-E′: mock-electroporated controls without ribonucleoproteins/HDR templates) (n = 4 biological replicates each in 1-3 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance (F) Changes in CAR-expression levels (MFI normalized to start) after target cell encounter. (TRAC and LV in 4 biological replicates; CD3ζ in 2 biological replicates). (G) Acute lymphoblastic leukemia xenograft mouse model using luciferase-labeled Nalm-6 (CD19+) tumor cells. 4 days post Nalm-6 administration, 1 × 106 cryopreserved, 14-day expanded TCR-deleted CAR+ T cells were injected systemically. Tumor burden was assessed via bioluminescence imaging. (n = 5-6; 2-way ANOVA with Geisser-Greenhouse correction of log-transformed bioluminescence imaging data followed by Holm-Šídák multiple comparison test, with individual variances computed for each comparison). Asterisks in this and all further figures represent different P values calculated in the respective statistical tests (not significant [ns], P > .05; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001).

Integration of a truncated CD19-specific CAR into CD3ζ, but not TRAC, conveys cytotoxicity in conventional T cells toward CD19+ leukemia cells. (A) full-length second-generation CAR protein (left) and virus-free knock-in strategies to integrate a full-length CAR into TRAC or a truncated CAR (truncCAR) into TRAC or CD3ζ. (B) Flow cytometry dot plots after knock-in. Transgene integration into TRAC or CD3ζ disrupts expression of the TCR/CD3 complex. (C) Relative cytotoxicity in coculture with (CD19+) Nalm-6 target cells and CD19 KO Nalm-6 control cells (VITAL assay). Calculation of relative cytotoxicity according to formula stated in methods section. (n = 2 biological replicates each in 2 technical replicates; ordinary one-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance). (D-G) Functional testing of CD3ζ truncCAR, T cells in comparison to TRAC CAR and LV CAR-T cells. (D) Mean fluorescence intensity (MFI) determined by flow cytometry as a measure of cellular CAR expression and normalized to each donor’s mean CAR MFI in the TRAC condition. (n = 7 biological replicates each in 2-5 technical replicates; mixed-effects analysis with Geisser-Greenhouse correction + Holm-Šídák multiple comparison test with individual variances computed for each comparison). (E) Relative cytotoxicity towards CD19+ cells assessed in a 6-hour VITAL assay. (mock-E′: mock-electroporated controls without ribonucleoproteins/HDR templates) (n = 4 biological replicates each in 1-3 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance (F) Changes in CAR-expression levels (MFI normalized to start) after target cell encounter. (TRAC and LV in 4 biological replicates; CD3ζ in 2 biological replicates). (G) Acute lymphoblastic leukemia xenograft mouse model using luciferase-labeled Nalm-6 (CD19+) tumor cells. 4 days post Nalm-6 administration, 1 × 106 cryopreserved, 14-day expanded TCR-deleted CAR+ T cells were injected systemically. Tumor burden was assessed via bioluminescence imaging. (n = 5-6; 2-way ANOVA with Geisser-Greenhouse correction of log-transformed bioluminescence imaging data followed by Holm-Šídák multiple comparison test, with individual variances computed for each comparison). Asterisks in this and all further figures represent different P values calculated in the respective statistical tests (not significant [ns], P > .05; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001).

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