Journal: Molecular Therapy Oncology
Article Title: Development of multivalent CAR T cells as dual immunotherapy and conditioning agents
doi: 10.1016/j.omton.2025.200944
Figure Lengend Snippet: Overview of ELECTRIC CAR (A) Schematic of ELECTRIC CARs. SCF is N terminus, followed by a (G 4 S) 3 linker, TPO, a second (G 4 S) 3 linker, FLT3LG, a CD28 hinge, transmembrane, and intracellular (IC) signaling domain, and CD3ζ. (B) ColabFold-predicted structure of extracellular domain of ELECTRIC CAR, with representative binding modes to the KIT, MPL, and FLT3. (C) Comparison of in vitro T cell expansion after viral transduction to Mock (non-transduced) T cells (two-way ANOVA; ∗∗∗∗ p < 0.0001). (D) Transduction efficiency (left) and ELECTRIC CAR MFI (right) of transduced CAR T cells 8 days after viral transduction. (E) Representative flow cytometry plot (left) of anti-hSCF detection by murine anti-IGG1-PE fluorescent antibody (right). (F–H) Nalm-6 (N6) B-ALL cells were transduced with a virus encoding the extracellular and transmembrane domains of KIT (F), MPL (G), and FLT3 (H). (I–K) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with GFP+ N6 cells overexpressing (OE) KIT (I), MPL (J), or FLT3 (K) and N6 GFP expression was measured over time via Incucyte Live Imaging. (L–N) ELECTRIC CAR T cells were co-cultured across a range of E:T ratios with N6 KIT OE (L), N6 MPL OE (M), and N6 FLT3 OE (N) and monitored via Incucyte Live Imaging. (O) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with a heterogeneous mixture of N6 KIT OE, N6 MPL OE, and N6 FLT3 OE (1:1:1 ratio of KIT:MPL:FLT3) and monitored via Incucyte Live Imaging. (P) Monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and ELECTRIC CARs were co-cultured with N6 KIT OE, N6 MPL OE, and N6 FLT3 OE at 1:1 E:T ratios for 24 h, and supernatants were harvested for cytokine production measurement by ELISA. Cytokine production was compared between Mock and CAR T cells for each type of target cell (two-way ANOVA, Tukey’s multiple comparison test). For I–P, data are mean ± s.e.m. of triplicate wells.
Article Snippet: Triplekine CARs were detected with an anti-human SCF antibody (Catalog #MAB655, R&D Systems, Minneapolis, MN) followed by an anti-IGG1 PE antibody (Clone: RMG1-1, BioLegend, San Diego, CA) or an anti-G 4 S Linker antibody (Clone: E702V, Cell Signaling Technology, Danvers, MA).
Techniques: Binding Assay, Comparison, In Vitro, Transduction, Flow Cytometry, Virus, Expressing, Cell Culture, Imaging, Enzyme-linked Immunosorbent Assay
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