ADCs against SEMA4A are potent and selective in vitro. (A) Fab-ZAP assay in NCI-H929 cells, MM1.S cells, and K562 cells, which do not express SEMA4A, as a control. Cells were incubated with Fab-ZAP alone or with clone 5E3 anti-SEMA4A or with an isotype control. Cell viability was measured by XTT assay at 72 hours. (B) Linker chemistry for 5E3-vedotin and 5E3-emtansine. (C) Dose–response curves for 5E3-vedotin and 5E3-emtansine in NCI-H929 and MM1.S cells. Citrate buffer was used as a control. Cell viability was measured by XTT assay at 72 hours. IC₅₀ values are specified, where the calculation was possible. NR = IC₅₀ was not reached. (D) NCI-H929 and MM1.S cells were treated with media only, citrate buffer (vehicle), 10 nM trastuzumab-vedotin (TRA-vedotin), 10 nM 5E3-vedotin, or 10 nM bortezomib (BTZ) and incubated with IncuCyte Caspase-3/7 apoptosis assay reagent for 72 hours and imaged by using an IncuCyte Live Cell Analysis system. (E) NCI-H929 cells were cocultured ± HS5 and treated with vehicle or dexamethasone (DEXA, 10 µM) for 72 hours. Total cell counts were determined by using Flow-Count Fluorospheres (Beckman Coulter) by flow cytometry and normalized to media-treated controls. (F) NCI-H929 were cocultured with HS5 and treated with either vehicle or 5E3-vedotin. Cell counts were determined as in panel E at 72 hours. CD138⁺ cells (G) or CD14⁺ cells (H) from myeloma patients were isolated by using microbeads (Miltenyi Biotec) and incubated with vehicle or 5E3-vedotin. Cell viability was measured by CellTiter-Glo or Annexin V and LIVE/DEAD staining by flow cytometry. Error bars indicate ±SD of a minimum of 3 replicates.