Fig. 2: OSCAR T cell characterization.

a Design of OSCAR CAR construct(top). Quantification of CD34 and mFab expression of OSCAR-1 and OSCAR-3 in primary T cells (bottom). Data are presented as percentage of CD34+ and mFab+ cells ± s.d. pooled from six donors (n = 6). b Quantification of OSCAR-1 and OSCAR-3 T cell expansion. Average of the absolute cell counts taken on day 0, 5 and 10 following CD3/CD28 bead expansion. Data pooled from three independent experiments on six donors (n = 6). Statistical comparisons performed by two-way ANOVA with Tukey’s multiple comparison test (ns not significant p > 0.05). c Cytotoxicity of OSCAR-1 and OSCAR-3 against ALPL-1 positive cells using a bioluminescence assay (BLI) measured at 6 h. CAR T cells from three donors (n = 3) and biological duplicates were used. Statistical comparisons performed using two-way ANOVA with Tukey’s multiple comparison test (ns not significant p > 0.05, ****p < 0.0001). d Cytotoxicity of OSCAR-1 and OSCAR-3 by BLI measured at 10 h at different E:T ratios (BL-41 negative control). Four independent experiments were pooled (n = 4). e Cytokine secretion upon overnight co-culture with ALPL positive cells. (n = 4 for all except IFN-y n = 2). SaOS-2 IFN-γ values were out of range for all separated experiments and were removed. Statistical comparisons obtained by two-tailed unpaired Student t-test (ns not significant p > 0.05, *p < 0.05, **p < 0.01). f Cytotoxicity of OSCAR-1 and OSCAR-3 upon repeated antigen stimulation (three). Data pooled from three donors (n = 3). Statistical comparisons performed with two-way ANOVA (ns not significant p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). g Proliferation of Cell Trace Violet (CTV)-labelled T cells (OSCARs,CD19CAR) upon repeated stimulation with ALPL positive targets (HEK-293^ALPL-1). Data pooled from four donors (n = 4). Statistical comparisons performed with two-tailed paired Student t-test (ns not significant p > 0.05, *p < 0.05, **p < 0.01). h Ca²⁺response of OSCARs upon contact with ALPL positive cells. Ca²⁺ levels are plotted against time (minutes) (left). Basal Ca²⁺ levels are indicated by a black line at “1.2” on the y-axis from mock T cells. Amplitude of Ca²⁺ responses (right) at the peak and at 10 min. i Quantification of Ca²⁺ responding OSCAR-1 and OSCAR-3 T cells. h, i, Data pooled from three experiments (n = 3). From 50 to 75 cells were counted per condition. p values obtained by two-tailed unpaired Student t-test (ns not significant p > 0.05, **p < 0.01). j Live cytotoxicity of OSCAR-1 and OSCAR-3 T cells against OHS spheroids using Incucyte. Cytotoxicity measured as loss of green fluorescence in GFP⁺ spheroids. Data are mean ± s.d. of sextuplicates. Data are from one representative experiment. Two independent experiments were performed (n = 2). k Cytotoxicity of OSCAR-1, OSCAR-3 on primary OS (OST-3 and OST-4) measured by xCELLigence at two E:T ratios (10:1; 5:1). Data pooled from three experiments (n = 3). Statistical comparisons were performed with one-way ANOVA test with Tukey’s post hoc test (ns not significant p > 0.05, ****p < 0.0001). For (a–h, and k–l), data are mean ± s.d.