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. 2024 Nov 18;15:9917. doi: 10.1038/s41467-024-53996-7

Fig. 3. Cotinine-labeled anti-CD40 scFv can be used as a dose-adjustable adapter for switchable Cot CAR-T cells in vitro.

Fig. 3

a Scheme of conventional CAR-T and switchable CAR-T cells. b Schematic diagram of the murine Cot CAR construct and a representative flow cytometry plot of Cot CAR expression on mouse T cells 4 days after transduction. c Chemical structure of carboxy cotinine (top left). Schematic diagram of cotinine-labeled anti-mouse CD40 scFv-Cκ (C1C02-Cot) (top right). Representative flow cytometry plot of binding of C1C02-Cot to A20 cells (bottom). d Cytotoxicity of Cot CAR-T cells against A20 cells preincubated with C1C02-Cot. e IFN-γ production after co-culture of Cot CAR-T cells with A20 cells preincubated with C1C02-Cot for 24 h, measured in triplicate. Data are presented as mean ± SEM. Statistical significance was determined by an unpaired two-tailed t test. Results are representative of three independent experiments (d, e). f CD40 expression levels in A20 cells and F4/80 (+) peritoneal macrophages (Mφ) as determined by staining with a commercially available anti-mouse CD40 antibody. g, h Comparison of dose-dependent cell binding affinity of C1C02-Cot between A20 and macrophage. Mean fluorescence intensities (MFIs) of binding are shown as values within the plot (g) and as a graph (h). i For toxicity readouts, macrophages were preincubated with various concentrations of C1C02-Cot and co-cultured with Cot CAR-T or CD40 CAR-T cells for 24 h. The amount of IL-6 in culture supernatants was measured (mean ± SEM, left axis and blue lines). For efficacy readouts, A20 cells (target) were preincubated with various concentrations of C1C02-Cot and then co-cultured with Cot CAR-T cells (effector) at an E:T ratio of 5:1 for 6 h. CD40 CAR-T cells were co-cultured with target cells not treated with C1C02-Cot. Percent cytotoxicity was calculated from flow cytometry-based viable cell counting (right axis and red lines). Results in g–i are representative of at least two independent experiments. p: p-value. Source data are provided in the Source Data file.