Fig. 1: Enhancing CAR-T cell efficacy by inducing Tn antigen expression.
From: Tuning CAR-T cells by targeting cancer-associated glycan in pancreatic cancer
a Biosynthetic pathway for O-glycans with polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) and C1GALT1 gene. b Schematic of mesothelin-targeting Chimeric Antigen Receptor (CAR)-T cells with glyco-bridge. c Graphic representation of the constructs used to make CAR-T cells with Tn-MUC1 bridge or CD19-bridge. The glyco-bridge contains a CD28 hinge and transmembrane domain (H/TM) and an inactive CD28 intracellular signaling domain (ICD). d Flow cytometry analysis of glyco-bridge surface levels on T cells using ALFA-Tag (left) and (G4S)3 linkers expression levels (right). e Schematic of the biosynthetic pathway for Tn antigen. f Flow cytometry analysis of Tn-MUC1 surface level on Capan-2 and Capan-2 C1GALT1 KO cells. g Representative real-time cytotoxicity assay against Capan-2 C1GALT1 KO cells at a 1:1 E:T ratio (relative to day 0 tumor seeding) from n = 3 distinct human blood cell donors. Results are mean ± s.d. of n = 3 independent measurements. h Surface level of Tn-MUC1 on Capan-2 following itraconazole treatment at the indicated concentrations for 48 h. i Representative real-time cytotoxicity assay against 2.5 µM itraconazole-treated Capan-2 cells at a 1:1 E:T ratio (relative to day 0 tumor seeding) from n = 3 three distinct human blood cell donors. Results are mean ± s.d. of n = 3 independent measurements. j Schematic of cell avidity measurement with acoustic force microscopy. k Strength of interaction between Capan2 C1GALT1 KO target cells and CAR-T cells expressing TnMUC1 or CD19-bridges. Percentage of total CAR-T cells remaining bound to target cells as the acoustic force ramp is applied from 0 to 1000 pN are shown. Results are mean ± s.e.m. of n = 3 independent measurements. l Percentage of CAR-T cells remaining bound to the target cells at the avidity plateau under 1000 pN force from Fig. 1k. Results are mean ± s.d. of n = 3 independent measurements. In d, f, h, representative flow cytometry data from three independent experiments. In g, i, and k, statistical analysis was performed by two-way ANOVA with correction for multiple comparisons. In l, statistical analysis was performed by one-way ANOVA with Tukey’s post hoc tests.
