Supplementary Material for: Enhancement of the Antitumor Effect on Combination Therapy of an Anticancer Drug and Its Antibody against Carcinoembryonic Antigen

<i>Background:</i> Carcinoembryonic antigen (CEA) is frequently overexpressed in various types of human cancers and is associated with cell adhesion. There are three possible mechanisms of cancer therapy that employ anti-CEA antibody (Ab): Ab-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or the prevention of CEA interaction with the extracellular matrix and/or intercellular adhesion molecules resulting in anoikis. In this study, the effect of C2-74, a human anti-CEA monoclonal Ab was evaluated. <i>Methods:</i> ADCC, CDC and anoikis assays in combination with C2-74 and an anticancer drug (5-fluorouracil or cisplatin) were investigated using tumor cell lines (MKN-45, MKN-74 and KATO III). In the anoikis assay, other human anti-CEA Abs and mouse anti-CEA-related cell adhesion molecule 6 Abs were also investigated using HLC-1 cells. <i>Results:</i> Additive cytotoxicity was observed when the anticancer drug and C2-74 on tumor cells were combined in the CDC assays, whereas in the anoikis assay, no such additive effect was observed. Anti-CEA-related cell adhesion molecule 6 Abs, but not anti-CEA Abs, accelerated anoikis in HLC-1 cells. <i>Conclusion:</i> A mechanism for the additive antitumor effect when an anticancer drug and C2-74 are combined is indicated mainly by CDC activity but is irrelevant to anoikis in tumor cells.