Linker stability assay

ADCs were incubated at a concentration of 150 μg/mL in mouse serum (Invitrogen) at 37°C in a shaking incubator. At various time point, aliquots were purified by affinity chromatography onto an antigen-coated resin, based on TnC A1 coupled to CNBr-activated Sepharose (GE Healthcare), washed with PBS and then eluted with 0.1 M glycine solution (pH = 3), prior to analysis by mass spectrometry.

For in-vivo analysis, ADCs were injected in the lateral vain of Balb/c nude mice at a dose of 10mg/Kg. After 24h or 48h, mice were sacrificed and blood was punctured from their heart and transferred into Heparin coated tubes. Tubes were centrifuged at 3000g for 5 min. Plasma was recovered and processed with the same method described above.