Generation of Defucosylated Anti-EpCAM Antibody

An anti-EpCAM mAb, EpMab-37, was established as previously described [27 (link)]. To switch the subclass of EpMab-37 from mouse IgG₁ to mouse IgG_2a (EpMab-37-mG_2a), we cloned V_H cDNA of EpMab-37 and C_H of mouse IgG_2a into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan). V_L cDNA of EpMab-37 and C_L cDNA of mouse kappa light chain were also cloned into the pCAG-Neo vector (Wako). To generate the defucosylated EpMab-37-mG_2a, the vector for the EpMab-37-mG_2a was transfected into FUT8 knockout ExpiCHO-S (BINDS-09) cells using the ExpiCHO Expression System (Thermo) [35 (link),36 (link),37 (link),38 (link),39 (link),40 (link),41 (link),42 (link),43 (link),44 (link),45 (link),46 (link),47 (link),48 (link),49 (link)]. Defucosytaled EpMab-37-mG_2a (EpMab-37-mG_2a-f) was purified using Ab-Capcher (ProteNova Co., Ltd., Kanagawa, Japan). Mouse IgG (cat. no. 140-09511) and IgG_2a (cat. no. M7769) were purchased from Wako and Sigma-Aldrich (St. Louis, MO, USA), respectively. A 281-mG_2a-f (a defucosylated anti-hamster podoplanin [PDPN] mAb, control mouse IgG_2a for ADCC reporter assay) was previously described [50 (link)]. Trastuzumab was purchased from the R&D systems (Minneapolis, MN, USA).