Extended Data Fig. 4: in vitro functionality analysis of Cilta-cel#4’s CARTs.
(a) Heatmap shows z-scores of the log2 fold changes for each marker across each row. Left: antigen-specific degranulation and cytokine production in CD4+ and CD8+ CARTs from peripheral blood (PB) and cerebrospinal fluid (CSF) from Cilta-cel#4 following coculture with MM.1S, compared to PB CARTs from three normal donors and Cilta-cel#22. Right: fold change of CD4+/CD8+ CART degranulation and cytokine production in the same samples. (b) In vitro cytotoxicity assay comparing Cilta-cel#4 PB CARTs at Day 13 with CARTs manufactured from three normal donors, using MM.1S as target. An effector: tumor ratio of 1:1 was used. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01. Exact P-values are provided in Supplementary Table S9. (c) In vitro cytotoxicity assay comparing Cilta-cel#4 PB and CSF CARTs at Month 2 (M2) with to Cilta-cel#22 PB CARTs, using RPMI-8226 as target. A range of effector: tumor ratios between 2:1 and 1:2 was used. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. Exact P-values are provided in Supplementary Table S9. (d) Serum cytokine profiling of Cilta-cel#4 from pre-lymphodepletion (Pre-LD), Day 7, peak expansion (Day 13), to Month 2. Left: heatmap shows z-score normalized cytokine concentration. Right, top: temporal kinetics of cytokines associated with T-cell proliferation (IL-1b, IL-2, IL-4, IL-7, IL-15, IL-18). Right, bottom: Temporal kinetics of cytokines implicated in Cytokine release syndrome (CRS; IL-6, TNF-α, GM-CSF, IL-8, IL-10). ^ represents cytokine concentration above upper limit of quantification of the LUMINEX assay. (e) CellTrace Violet-stained CARTs from three normal donors were seeded into 96-well plates, stimulated with IL-2: 10 ng/mL, IL-7: 20 g/mL, or IL-15: 20 ng/mL, and incubated for five days. Left: proliferation was assessed by flow cytometry analysis of CellTrace Violet dilution. Right: phosphorylated STAT5 (pSTAT5+) activation following 15 min of activation with IL-2 (10 ng/mL), IL-7 (20 ng/mL), or IL-15 (20 ng/mL) in 3 normal donors. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. (f) Baseline IL2, IL7, and IL15 receptor-alpha (RA) chain expression in Cilta-cel#4 compared to 3 normal donors before cytokine stimulation. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Exact P-values are provided in Supplementary Table S9. (g) CCR5 median fluorescence intensity (MFI) in three normal donors and Cilta-cel#4 following cytokine stimulation with IL-2 (10 ng/mL), IL-7 (20 ng/mL), or IL-15 (20 ng/mL). Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Exact P-values are provided in Supplementary Table S9. (h) Absolute count (left) and viability (right) of CD3+ T-cells from 3 normal donors after 3 days of CD3/28 stimulation in the presence of increasing maraviroc concentrations (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM). Data are shown as mean values with error bars representing the standard error of the mean (SEM) from 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. CSF: cerebrospinal fluid; E:T: effector: tumor ratio.
