Fig. 4: Construction of highly specific and effective GD2-B7H3 T cells.

a Schematic of SynNotch receptor with extracellular ligand-binding domain (scFv) directed against a TAA. Upon ligand recognition by the SynNotch receptor, an orthogonal transcription factor is cleaved from the cytoplasmic tail that activates a custom genetic construct. b Schematic structure of activation of the BFP reporter gene when the high-affinity scFv GD2^E101K SynNotch receptor is presented with GD2 TAA. c, upper: Representative dot plot of BFP expression by flow cytometry in GD2-BFP T cells co-cultured with NBL cells for 24h. Lower: Time-course kinetics of BFP expression by flow cytometry in GD2-BFP T cells with and without NBL co-culture for 3 days. d Histograms of BFP expression in GD2-BFP T cells co-cultured with GD2⁺ CHLA255 and GD2⁻ LAN6 NBL cells from 0 to 48 h. e Schematic structure of B7H3 CAR activation when the high-affinity scFv GD2^E101K SynNotch receptor is presented with GD2 TAA and subsequent expression of B7H3 CAR and resultant cytotoxic activity. f Histogram representing the activation marker (CD107a) on GD2-B7H3 cells co-cultured with GD2⁺ NBL cell line CHLA255, GD2⁻ LAN6, or PMA/Ionomycin (positive control) after 24h. g Expansion of UT, GD2-B7H3 T cells co-cultured for 5 days with CHLA255 NBL cells at various effector:target ratios. h Summary of cytokine release of GM-CSF, IFNγ, IL2, MCP1, and TNF measured by ELISA from UT and GD2-B7H3 T cells co-cultured with CHLA255 NBL cells at 1:1 effector:target ratio i kinetics of cytotoxicity of UT and GD2-B7H3 T using live-cell imaging (enumeration of GFP⁺ tumor cells) against CHLA255 NBL cells at indicated E:T ratios. j Representative bioluminescence images of CHLA255 NBL tumor growth upon i.v. injection into NSG mice (1 × 10⁶ cells/mouse) and treatment 7 days later with i.v. injection of 1×10⁷ UT, GD2-B7H3, or CD19-B7H3 T cells. Surviving mice were followed for a minimum of 100 days post-tumor inoculation. Data shown are representative of three (c, d, f, g) and four (h) independent experiments. minimum of four replicate (i), mean ± SD (c, g, h, i). Two-tailed t test (c, g, h). n=5 mice (GD2-B7H3, CD19-B7H3, and UT). Individual BLI and source data are provided as a Source Data file.