DAR2-ARC-IgG and DAR4-ARC-IgG at above 10 μM stock concentrations were combined with the drug-linker conjugate (2′-Cl-araNAD+-MMAF) in Tris buffer (50 mM Tris, pH 8.5) at a final molar concentration ratio of 1:200 (antibody : drug-linker conjugate) on ice for 1 hour. Then, buffer exchange was performed using PBS and filters with 30 kDa cut-off (MilliporeSigma, MA, USA) to remove free drug-linker conjugate.
To generate ADC surrogates with Alexa Fluor 488 or FITC as the payload, the linker 2′-Cl-araNAD+-N3 was first conjugated to DAR2-ARC-IgG or DAR4-ARC-IgG with the same method described above. Then, acquired fusion antibody-linkers were incubated with Alexa Fluor 488 DBCO (Click Chemistry Tools, AZ, USA) or DBCO-PEG3-FITC (CONJU-PROBE, CA, USA) in PBS buffer (pH 7.4) at a molar ratio of 1:50 (antibody-linker : fluorescent dye) for 30 minutes on ice. Finally, buffer exchange was performed using PBS (pH 7.4) and filters with 30 kDa cut-off to remove free surrogate payloads. The concentrations of generated ADCs and ADCs surrogates were determined by Bradford assay kits (Thermo Fisher Scientific, MA, USA).
To generate ADC surrogates with Alexa Fluor 488 or FITC as the payload, the linker 2′-Cl-araNAD+-N3 was first conjugated to DAR2-ARC-IgG or DAR4-ARC-IgG with the same method described above. Then, acquired fusion antibody-linkers were incubated with Alexa Fluor 488 DBCO (Click Chemistry Tools, AZ, USA) or DBCO-PEG3-FITC (CONJU-PROBE, CA, USA) in PBS buffer (pH 7.4) at a molar ratio of 1:50 (antibody-linker : fluorescent dye) for 30 minutes on ice. Finally, buffer exchange was performed using PBS (pH 7.4) and filters with 30 kDa cut-off to remove free surrogate payloads. The concentrations of generated ADCs and ADCs surrogates were determined by Bradford assay kits (Thermo Fisher Scientific, MA, USA).