Dog igg

The anti-hEGFR mAb, EMab-134, was developed as previously described [14 (link)]. To produce E134B, we subcloned the V_H cDNA of EMab-134 and C_H cDNA of Dog IgGB into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), along with the V_L cDNA of EMab-134 and C_L cDNA of dog kappa light chain into the pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation). Two vectors of E134B were transfected into BINDS-09 cells (FUT8-deficient ExpiCHO-S cells) using the ExpiCHO Expression System (Thermo Fisher Scientific Inc., Waltham, MA, USA) [16 (link)]. The resulting mAb, E134Bf, was purified with Protein G-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA, USA) [16 (link)]. Dog IgG was purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA).

Previously, we have established mouse-dog chimeric anti-EGFR mAb (E134B) [26 (link)] and anti-HER2 mAb (H77B) [27 (link)]. In this study, to generate E134Bf-H77scFv, we first constructed a single chain Fv of H77B (H77scFv) by connecting the V_H and V_L cDNA of H77B with a linker sequence (GGGGSGGGGSGGGGS). The H77scFv cDNA was further fused at the 3′ end of the light chain cDNA of E134B. The cDNA of the E134B heavy chain and E134B light chain-H77scFv were transduced into BINDS-09 (FUT8 knockout ExpiCHO-S) cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.) to produce the defucosylated form [25 (link),26 (link),27 (link),28 (link),29 (link),30 (link),31 (link),32 (link)]. A defucosylated and bispecific antibody, E134Bf-H77scFv, was purified using Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan). We confirmed their purity by SDS-PAGE (Supplemental Figure S1). Dog IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

The canine melanoma cell lines CMeC, LMeC, CMM-1, and CMM-2^{32 (link),33 (link)}, were cultured as described previously^{6 (link)}. The canine osteosarcoma cell lines POS^{34 (link)} and HMPOS^{35 (link)} were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 200 μg/mL streptomycin, and 200 U/mL penicillin (Thermo Fisher Scientific) at 37 °C under a 5% CO₂ atmosphere. Canine PBMCs were purified from heparinized blood obtained from healthy beagles by density gradient centrifugation on Percoll (GE Healthcare UK, Buckinghamshire, UK) and cultured as described previously^{6 (link)}. PBMCs were stimulated with 5 μg/mL Staphylococcal Enterotoxin B (SEB) (Sigma-Aldrich) and 1 μg/mL anti-canine CD28 antibody (eBioscience, San Diego, CA). In some experiments, cells were co-treated with 5 μM meloxicam (Sigma-Aldrich), 2.5 μM Prostaglandin E2 (Cayman Chemical), and/or 20 μg/mL canine chimeric anti-PD-L1 antibody c4G12^{11 (link)} as indicated. The same concentrations of DMSO (Nacalai Tesque, Kyoto, Japan) and dog IgG (Jackson ImmunoResearch, West Grove, PA) were used as negative controls.