LY6G6D-TDB activity is target dependent. A, Killing of engineered LY6G6D-expressing 293 cells and parental cells. Forty-eight-hour time point. B, Killing of HT55 cells when co-cultured with either total PBMCs, or CD3+ T-cell–depleted PBMCs in the presence of 1G4/CD3H. 48-hour time point. C and D, CD8+ and CD4+ T-cell activation in co-culture of HT55 cells and PBMCs in the presence of either 1G4/CD3H, or a control TDB, or culture of PBMCs alone in the presence of 1G4/CD3H. 48-hour time point. E, Cytokine in culture media of co-culture of HT55 cells and PBMCs or culture media of PBMCs alone in the presence of 1G4/CD3H. 24-hour time point. F, Intracellular level of granzyme B in CD8+ T and CD4+ T cells in co-culture of PBMCs and HT55 in the presence of 1G4/CD3H at 24-, 48-, and 72-hour time points, measured by flow cytometry. G, Intracellular level of Ki67 in CD8+ T and CD4+ T cells in co-culture of PBMCs and HT55 in the presence of 1G4/CD3H at 24-, 48-, and 72-hour time points, measured by flow cytometry. Representative data of two to three donors.