Extended Data Fig. 6: Construction and characterization of β-Lac-expressing murine SEAKER cells.

(a) SEAKER construct encoding secreted β-Lac and a murine CAR: β-Lac-MUC28z (β-Lac/α-MUC16/CD28/CD3ζ). LTR = long terminal, Ψ = psi packaging element, FLAG = FLAG epitope tag (pink), P2A = 2A self cleaving peptide, α-MUC16 scFv = MUC-16-specific mouse-derived single chain variable fragment, myc = Myc epitope tag (brown), mCD28 = mouse CD28 costimulatory domain (green), mCD3ζ = mouse CD3 zeta chain (red). (b) Flow cytometry analysis of α-MUC16 CAR expression in retrovirally-transduced primary mouse T cells (fluorescently (phycoerythrin, PE) labeled anti-idiotype antibody; representative data from 5 independent experiments). (c) Trans-cytotoxicity of supernatant fluid (sn) from β-Lac-MUC28z SEAKER cells with or without Ceph-AMS (3: 107 nM) against mouse EL4 lymphoma cells, compared to prodrug alone and parent drug AMS (1: 500 nM) (24 h, CellTiter-Glo assay; mean ± s.d. of n = 3 technical replicates; Student’s two-tailed t-test: ns = not significant, ***p < 0.001; experiment was perfomed once).