(A) Preparation and electrospray ionization mass spectrometry (ESI-MS) analysis of homogeneous ADC 1. Top panel: N88A/N297A anti-EGFR mAb (cetuximab mutant). Middle panel: mAb-linker conjugate. Bottom panel: homogeneous ADC 1. Asterisk (*) indicates a fragment ion detected in ESI-MS analysis.
(B) Preparation and HIC analysis of Cys conjugate 2.
(C) Preparation and ESI-MS analysis of Lys conjugate 3.
(D and E) Cell-based ELISA in U87ΔEGFR-luc (EGFRvIII+) and HEK293 (EGFR−, EGFRvIII−) cells.
(F–H) Cell-killing potency in U87ΔEGFR-luc, Gli36δEGFR (EGFRvIII+), and HEK293 (mean values ± SEM, n = 3). Concentrations are based on the antibody dose without normalizing to each DAR.