Fig. 2: Binding and in vitro activity of HER2-XPAT protein, its partially unmasked metabolites and unmasked HER2-TCE.
a, Binding affinities to human HER2 and CD3 at 37 °C by surface plasmon resonance. Data are KD (n = 12 technical replicates of a single experiment for HER2-XPAT protein and HER2(1x-N); n = 8 for HER2(1x-C) and uTCE). Surface plasmon resonance sensorgrams for these data are provided in Supplementary Figs. 3–10. b–d, In vitro tumor cytotoxicity of HER2-XPAT protein and its metabolites following a 48-h incubation with co-cultures of huPBMCs and the high HER2-expressing human tumor cell lines (1:1 effector–target ratio) SKOV3 (b), BT-474 (c) or the medium-low HER2-expressing MCF7 cell line (d). e, In vitro cytotoxicity of HER2-XPAT protein and its metabolites against BT-474 cells co-cultured with huPBMCs (1:1 effector–target ratio). f, Impact of the protease-cleavable linker on in vitro cytotoxicity versus BT-474 cells co-cultured with huPBMCs. g,h, CD69-positive T cells (g) and IL-2 secretion (h) following 72-h incubation of huPBMC/SKOV3 co-cultures with HER2-XPAT protein or its unmasked form (uTCE). i, Target-dependent T-cell activation with HER2-XPAT protein and its metabolites. CD3-expressing Jurkat reporter T cells were incubated with BT-474 cells at a 5:1 effector–target ratio for 6 h, followed by quantification of NFAT-induced luciferase activity and measured in relative luminescence units (RLUs). Mean data for n = 2 technical replicates within one single experiment (b–i). Extended Data Table 1 provides a summary of EC50 values for the different forms of the HER2-XPAT proteins in the cytotoxicity and reporter T-cell activation assays.
