pWPXL-HF2AL-expressed zaptuximab exhibited a better light chain/heavy chain balance than did zaptuximab expressed from pWPXL-LF2AH. (a) Schematic illustration of the full-length chimeric antibody expression cassette using the furin/2A sequence. (b) Expression of zaptuximab in supernatants from pWPXL-HF2AL or pWPXL-LF2AH-transfected HEK 293 cells. The samples were separated by SDS-PAGE under reducing conditions and probed with a goat anti-human IgG (H+L) polyclonal antibody. The data presented are representative of three independent experiments. (c) Cytotoxicity of zaptuximab expressed by pWPXL-HF2AL or pWPXL-LF2AH in tumor cells. HCT116 and SVT35 Jurkat T cell lines were treated with medium from HEK 293 cells transiently transfected with pWPXL-HF2AL or pWPXL-LF2AH for 24 hours. Cell viability was determined by MTS assays (mean ± SD). Replicates were averaged, and the error bars represent the SEM of three independent experiments. H, heavy chain; L, light chain; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SP, signal peptide.