Figure 5. CD3ζ -editing enables... | American Society of Hematology

Figure 5.

$CD3ζ-editing enables redirection of NK cells with CARs and does not impede canonical NK cell functions in vitro. CAR editing in primary NK cells via LV CAR transfer, TRAC-CAR or CD3ζ-truncCAR-integration: (A) CAR+ frequencies after editing (n = 6 biological replicates, mixed-effects analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparison test with individual variances computed for each comparison). (B) mean CAR expression (MFI) normalized to CD3ζ-truncCAR integrated NK cells and robust coefficient of variation in CAR+ cells (n = 6 biological replicates; t test). (C) CAR-dependent cytotoxicity detected in a VITAL assay (data normalized to mock-electroporated (wildtype) NK cells; n = 6 biological replicates each in 3-4 technical replicates; 2-way ANOVA followed by Tukey’s multiple comparison test with a single pooled variance). (D) Degranulation as indicator of NK effector function via flow cytometric detection of CD107a (n = 6 biological replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance). (E) ADCC of primary (CAR) NK cells against CD20+ bGal− Jeko-1 cells. Bars represent killing for each condition in the presence of a CD20-targeting monoclonal antibody (0.5 μg/mL) normalized to the respective condition without supplemented antibody (n = 5 biological replicates; mixed-effects analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparison test with individual variances computed for each comparison). (F-H) CD19-CAR (2) transfer to NK-92 cells via AAVS1 integration of a CMV promotor-controlled, full-length CAR or CD3ζ integration of a truncCAR. CAR+ fractions were enriched using MACS. (F) CAR expression in flow cytometry histograms. (G) CAR-dependent cytotoxicity in a 4-hour VITAL-assay (n = 6 technical replicates; two-way ANOVA with Tukey’s multiple comparison test with a single pooled variance. (H) CAR-independent cytotoxicity towards the MHC I deficient, CD19− K562 (control) cell line (n = 15 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance).$

CD3ζ-editing enables redirection of NK cells with CARs and does not impede canonical NK cell functions in vitro. CAR editing in primary NK cells via LV CAR transfer, TRAC-CAR or CD3ζ-truncCAR-integration: (A) CAR⁺ frequencies after editing (n = 6 biological replicates, mixed-effects analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparison test with individual variances computed for each comparison). (B) mean CAR expression (MFI) normalized to CD3ζ-truncCAR integrated NK cells and robust coefficient of variation in CAR⁺ cells (n = 6 biological replicates; t test). (C) CAR-dependent cytotoxicity detected in a VITAL assay (data normalized to mock-electroporated (wildtype) NK cells; n = 6 biological replicates each in 3-4 technical replicates; 2-way ANOVA followed by Tukey’s multiple comparison test with a single pooled variance). (D) Degranulation as indicator of NK effector function via flow cytometric detection of CD107a (n = 6 biological replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance). (E) ADCC of primary (CAR) NK cells against CD20⁺ bGal⁻ Jeko-1 cells. Bars represent killing for each condition in the presence of a CD20-targeting monoclonal antibody (0.5 μg/mL) normalized to the respective condition without supplemented antibody (n = 5 biological replicates; mixed-effects analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparison test with individual variances computed for each comparison). (F-H) CD19-CAR (2) transfer to NK-92 cells via AAVS1 integration of a CMV promotor-controlled, full-length CAR or CD3ζ integration of a truncCAR. CAR⁺ fractions were enriched using MACS. (F) CAR expression in flow cytometry histograms. (G) CAR-dependent cytotoxicity in a 4-hour VITAL-assay (n = 6 technical replicates; two-way ANOVA with Tukey’s multiple comparison test with a single pooled variance. (H) CAR-independent cytotoxicity towards the MHC I deficient, CD19⁻ K562 (control) cell line (n = 15 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance).

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