Extended Data Fig. 5: Cilta-cel and Ide-cel in vitro functional differences.
(a) Overlay histogram of tNGFR and BCMA-CAR expression in SUP-T1 cell line stably transduced with ide-cel CAR (ide-cel), cilta-cel CAR (cilta-cel), mesothelin-directed CAR (M5) or untransduced control cells (UTD). (b) Comparison of BCMA-binding avidity between ide-cel vs cilta-cel, with M5 and UTD as controls. Left: Percentage of MM.1S cells bound across a range of applied forces. Right: Percentage of MM.1S binding at 1000 pN force; data are presented as mean values ± the standard error of the mean (SEM) from 2–5 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. (c) Overlay histogram of tNGFR and VHH-CAR expression in Jurkat triple parameter reporter (Jurkat_TPR) cell line stably transduced with the ScFv-based ide-cel CAR (ide-cel) or VHH-based cilta-cel CAR (cilta-cel). (d) Baseline (tonic) NFAT and NKFB activation status in Jurkat_TPR cells expressing ide-cel and cilta-cel in the absence of antigen stimulation; data are presented as mean values ± 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. (e) Left: fold-change from unstimulated in NFAT and NKFB % positive cells (top left) and median fluorescence intensity (MFI, bottom left) in Jurkat_TPR expressing ide-cel or cilta-cel following 18 and 24 h of stimulation with MM.1S with a range of effector: tumor ratios between 5:1 to 1:5. Right: fold-change from unstimulated in NFAT and NKFB % positive cells (top right) and median fluorescence intensity (MFI, bottom right) in Jurkat_TPR expressing ide-cel or cilta-cel following 1:1 stimulation with MM.1S across multiple timepoints ranging from 3 to 72 h. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from 3 biological replicates. (f) Jurkat_TPR expressing ide-cel or cilta-cel were co-cultured 1:1 with irradiated MM.1S for 24 to 48 h, then separated from MM.1S using tNGFR-PE magnetic bead (positive) selection. The contour plot confirms high post-sort purity of Jurkat_TPR cells used for subsequent Seahorse metabolic analysis. (g) Mitochondrial respiration, (h) Maximal and spare respiratory capacity, (i) Glycolytic rate, (j) Total ATP production (bioenergetic capacity), and (k) ATP production rate in Jurkat_TPR expressing ide-cel or cilta-cel assessed at baseline (unstimulated), and after 24 and 48 h of stimulation with MM.1S. Data are presented as mean values ± the standard error of the mean (SEM) from 3–6 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. APC: allophycocyanin; ATP: adenosine triphosphate; ECAR: extracellular Acidification Rate; MFI: median fluorescence intensity; NFAT: nuclear factor of activated T-cells; NFkB: nuclear factor kappa-light-chain-enhancer of activated B-cells; NS: not significant; OCR: oxygen consumption Rate; PE: phycoerythrin; ScFV: single-chain variable fragment; SRC: spare respiratory capacity; tNGFR: truncated nerve growth factor receptor; VHH: camelid single-domain antibody/nanobody.
