Fig. 1

SS1 CAR T-cells with varying endodomains exhibit differing immunophenotypes. A Schematic of 4-1BBζ, CD28ζ and Mutζ SS1 CAR constructs expressed via Moloney murine leukaemia retroviral vectors. SP: Signal peptide; scFv: Single chain variable fragment; TM: Transmembrane; ICD: Intracellular domain. Magenta and teal strips denote functional and non-functional immunoreceptor tyrosine-based activation motifs (ITAMs) respectively: Y: tyrosine; F: phenylalanine residues. B, C Transduction efficiency as measured by Protein L of B CD4+, and C CD8+ T-cell products. D, E CAR expression in D CD4+, and E CD8+ CAR T-cells measured by biotinylated protein L and streptavidin-APC. F The CD4: CD8 ratio of each cell product; UT: untransduced. G Stacked bar graphs of mean memory cell composition (totaling 100%) in CD4+ and CD8+ cell products phenotyped with anti-CCR7 and anti-CD45RO antibodies. In B–G, the mean and standard error of the mean (SEM) are indicated. Values are presented as averages of 3 donors and therefore the total values differ subtlety. H, I Expression of exhaustion markers TIM3 and PD1 in H CD4+, and I CD8+ CAR T-cell products; the mean is indicated. Experiments were performed with n = 3 independent donors, each analysed on different days, in B–F and H, I; each donor is distinguished by a unique symbol. Comparisons were made between all cell products by one-way ANOVA with Tukey’s correction for multiple comparisons; only statistically significant differences are indicated (*p < 0.05, **p < 0.01, ***p < 0.001). J, K xCELLigence real-time cytotoxicity assay of SS1 cell products against J Capan2, and K AsPC1 MSLN KO at a normalized effector to target ratio of 2:1. Lysis is normalized to untransduced T-cells and data are representative of 3 independent experiments with independent donors performed on different days. In J, K the mean and standard deviation are indicated