Pcag ble vector

PMab-38, a mouse anti-dPDPN mAb was developed in our previous study.^{(11 (link))} To develop the mouse–canine chimeric antibody P38B, V_H of PMab-38 and C_H of canine immunoglobulin G (IgG) subclass B were subcloned into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and V_L of PMab-38 and C_L of canine IgG were subcloned into the pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation).^{(16 (link))} Using the ExpiFectamine CHO Transfection kit (Thermo Fisher Scientific, Inc., Waltham, MA), the expression vectors were transfected into ExpiCHO-S cells to express P38B antibody. P38B was purified using Protein G-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA).

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).

CHO-K1 and P3X63Ag8U.1 (P3U1) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The horse kidney cell line, FHK-Tcl3.1, was established at Yamaguchi University [33 (link)]. The horPDPN bearing an N-terminal PA16 tag (PA16-horPDPN) was inserted into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) [32 ]. The PA16 tag comprises 16 amino acids (GLEGGVAMPGAEDDVV) [34 (link)]. CHO-K1 cells were transfected with pCAG-Ble/PA16-horPDPN using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). Stable transfectants were selected by limiting dilution and cultivated in a medium containing 0.5 mg/mL of zeocin (InvivoGen, San Diego, CA, USA).
P3U1, CHO-K1, and CHO/horPDPN cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and FHK-Tcl3.1 was cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.) [32 ]. All media were supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.). Cells were grown at 37 °C in a humidified environment with an atmosphere of 5% CO₂ and 95% ambient air.

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).

Chinese hamster ovary (CHO)-K1 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). In our previous studies, we inserted dPDPN with an N-terminal PA tag and a C-terminal RAP tag-MAP tag (PA-dPDPN-RAP-MAP) in a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).^{(10 (link))} The PA tag,^{(16 (link))} RAP tag,^{(17 (link))} and MAP tag^{(18 (link))} consist of 12 amino acids each, namely, GVAMPGAEDDVV, DMVNPGLEDRIE, and GDGMVPPGIEDK, respectively. CHO-K1 cells were transfected with pCAG-Ble/PA-dPDPN-RAP-MAP using Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA) resulting in the cell line CHO/dPDPN. CHO-K1 and CHO/dPDPN were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

The Genome Network Project clone IRAK021G03 (EpCAM) was provided by the RIKEN BioResource Research Center through the National BioResource Project of the MEXT and AMED agencies of Japan. EpCAM cDNA plus a C-terminal PA tag that is recognized by the anti-PA tag mAb (NZ-1), was subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). N-terminal PA-tagged EpCAM deletion mutants (dN44, dN64, dN84, dN104, dN124, dN144, dN164, dN184, dN204, dN224, and dN244) were produced using a HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany), and subcloned into the pCAG-Ble vector.

We previously inserted dPDPN with an N-terminal PA tag (GVAMPGAEDDVV)^{(19 (link))} and a C-terminal RAP tag (DMVNPGLEDRIE)^{(20 (link))}-MAP tag (GDGMVPPGIEDK)^{(21 (link))} (PA-dPDPN-RAP-MAP) in a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation).^{(11 (link))} CHO-K1 cells (American Type Culture Collection, Manassas, VA) were transfected with pCAG-Ble/PA-dPDPN-RAP-MAP using Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA) for developing CHO/dPDPN.
CHO/dPDPN and CHO-K1 cells were cultured using RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.). Antibiotics, such as 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B (Nacalai Tesque, Inc.) were added into the medium. Cells were cultivated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.