Vcmmae

Cell lines (Raji, Daudi, OCI-AML3, OVCAR-3, HCT116) were maintained in RPMI media supplemented with 10% (v/v) cosmic serum (GE Healthcare) and 1% (v/v) Penicillin–Streptomycin (GE Healthcare). Luciferase-expressing Raji cells (Rajiluc) were generated by stably transfecting Raji cells using the plasmid pLenti-CMV V5-luciferase (Addgene) using methods as previously reported.^{26 (link)} PBMCs were isolated from healthy donor blood obtained from the Hematopoietic Biorepository and Cellular Therapy Core at CWRU and maintained in RPMI media supplemented with 10% (v/v) cosmic serum (GE Healthcare) and 100 U mL⁻¹ Penicillin with 100 μg mL⁻¹ Streptomycin (GE Healthcare). Monomethyl auristatin E (MMAE) and vcMMAE were obtained from MedChemExpress, (Monmouth Junction, NJ).

vcMMAE (MedChem Express) was conjugated to PVX via the sulfhydryl side chains on the cysteine residues using the maleimide chemistry. 7500 molar excess of vcMMAE was reacted overnight with PVX at a protein concentration of 1 mg mL⁻¹ in presence of 10% (v/v) DMSO. PVX–vcMMAE was purified via ultracentrifugation at 112 000g for 3 hours over 30% sucrose cushion to remove unbound vcMMAE. The recovered PVX–vcMMAE pellet was resuspended in 0.1 M potassium phosphate buffer pH 7 and dialyzed against 0.1 M phosphate buffer. Purified PVX–vcMMAE were imaged by transmission electron microscopy TEM (FEI Tecnai Spirit G2 BioTWIN Transmission Electron Microscope) for particle stability and analyzed by dynamic light scattering (DLS) to rule out particle aggregation. Drug loading was quantified by SDS-PAGE (4–12% NuPAGE gels, 1× MOPS running buffer, Invitrogen) followed by densitometry analysis performed using the band analysis tool in ImageJ software (https://imagej.net/Fiji).