Generation of Anti-PODXL Antibodies

PcMab-47, a mouse anti-PODXL mAb (IgG₁, kappa), was developed as previously described [17 (link)]. The mouse IgG was purchased from Sigma-Aldrich Corp. (St. Louis, MO). To generate 60-mG_2a, appropriate V_H cDNA of PcMab-60 and C_H of mouse IgG_2a were subcloned into pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and V_L and C_L cDNAs of PcMab-60 were subcloned into pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation). To generate 60-mG_2a, antibody expression vectors were transfected into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher Scientific). To generate 60-mG_2a-f, antibody expression vectors were also transfected into BINDS-09 (FUT8-knocked out ExpiCHO-S cells) using the ExpiCHO Expression System [23 (link)]. PcMab-60, 60-mG_2a, and 60-mG_2a-f were purified using Protein G-Sepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA).