In vitro and in vivo cytotoxicity of MSLN-targeted ADCs in MSLN⁺leukemia cell lines. (A) In vitro cytotoxicity of AR in MV4;11-MSLN⁺ cell lines and IC₅₀ values. Controls are IC-AR and treatment of the parental (MSLN⁻) lines. In vitro cytotoxicity of the ADC anti-MSLN–DGN462 with an indolino-benzodiazepine dimer payload in MV4;11-MSLN⁺ and MV4;11 parental cells (B) and Nomo-1 parental cells and Nomo-1–MSLN^KO cells (C). (D) Kaplan-Meier survival plots of K562-MSLN⁺ cell–xenografted mice treated with AR compared with IC-AR, chemotherapy (Chemo), and no treatment. (E) Kaplan-Meier survival plots of MV4;11-MSLN⁺ xenografted mice treated with AR, along with controls: IC-AR (IC) and untreated. (F) PB leukemia burden was assessed in MV4;11-MSLN⁺ mice by flow cytometry. (G) Treatment of MSLN⁺ PDX NTPL-146 with AR resulted in a dose-dependent improvement in median survival with respect to untreated mice. Mice treated with AR vs IC-AR for 2 cycles (dashed lines; n = 5 per group) experienced a median survival of 82 days vs 32 days (P = .0018) and mice treated for 3 cycles (solid lines; n = 4 per group) had a median survival of 132 days vs 33 days, respectively (P = .0069; n = 4 per group). (H) Treatment of the MSLN⁻ PDX DF-2 with AR did not demonstrate any target-dependent efficacy compared with untreated IC-AR mice. Mice treated with AR vs IC-AR for 1 cycle (dotted lines; n = 4 per group) experienced an identical median survival of 4 days (P = 1.0) and mice treated for 2 cycles (solid lines; n = 5 per group) had identical median survival of 12 days, respectively (P = .173; n = 4 per group). (I) Quantification of cell surface mesothelin expression using BD Quantibrite, as measured by antibodies bound per cell in the MSLN⁺ ovarian cancer cell line OCVAR-3 used as positive control and the PDX models NTPL-146 and DF2. ND, IC₅₀ could not be determined with 95% CIs.