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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) <t>BCMA</t> expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.
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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) <t>BCMA</t> expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.
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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) <t>BCMA</t> expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.
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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) <t>BCMA</t> expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.
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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) <t>BCMA</t> expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.
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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) <t>BCMA</t> expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.
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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) <t>BCMA</t> expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.
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Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) BCMA expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.

Journal: Frontiers in Immunology

Article Title: Tailored strategies for improved control of CAR-T cells in multiple myeloma

doi: 10.3389/fimmu.2026.1740345

Figure Lengend Snippet: Efficiency of ADCC and ADC on SLAMF7 and CD19 CAR-T cells in vitro ; (A) Schematic depiction of EGFR-specific ADCC; (B, C) CD19 and SLAMF7 CAR-T cells (CD4:CD8 1:1) were incubated with EGFR antibody cetuximab [50 µg/ml] and cocultured with effector cells (CFSE stained, PBMC w./w.o. NK cells, E:T 50:1 and NK cells, E:T 10:1) for 24 h, CAR-T cell elimination through ADCC is dependent on NK cell availability and CAR-T cell construct, Representative Flow cytometry plots (B) and statistical analysis ((C) n=3) are shown; (D) Lymphocyte (n=12) and NK cell (n=8) counts are reduced after lymphodepleting chemotherapy (d-5, -4 and -3) and only gradually recover; (E) Schematic depiction of the ADC assay; (F) BCMA expression of targeted cells via flow cytometry, (G, H) Cells expressing BCMA or not, were incubated with BCMA targeting ADC belantamab-mafodotin [50 µg/ml]; SLAMF7 CAR-T cells, UTD ctrl T cells (72 h; (G) ) and the human MM cell line OPM-2 (48 h & 72 h; (H) ) are shown; Control antibody: Human IgG1 Isotype Control; n=3 independent donors; Each experiment was performed in triplicates; Two-way ANOVA statistic test was performed using GraphPad Prism 9; Abbreviations: ADC, Antibody-drug conjugate; ADCC, Antibody-dependent cell cytotoxicity; ALC, Absolute Lymphocyte Count; CAR, Chimeric antigen receptor; Ctrl, Control; MM, Multiple myeloma; SLAMF7, SLAM Family member 7 (CD319); UTD, Untransduced. **** = P ≤ 0,0001; *** = P ≤ 0,001; * = P ≤ 0,05.

Article Snippet: Antibodies used in this research report were specific for BCMA-Antibodies used in this research report were specific for BCMA (Miltenyi Biotec; 130-119-152), EGFR (Cetuximab, in-house labelled), CD8 (Miltenyi Biotec; 130-110-683) and CD4 (Miltenyi Biotec; 130-114-534).

Techniques: In Vitro, Incubation, Staining, Construct, Flow Cytometry, Expressing, Control