Pcag neo 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).

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).

CHO-K1 cells were obtained from the American Type Culture Collection and grown in Ham’s F12 Nutrient Mixture medium supplemented with penicillin, streptomycin, and 10% fetal bovine serum at 37 °C with 5% CO₂. FLAG-tag (N-DYKDDDDK-C)-fused human PSA (kallikrein-3, KLK3) cDNA was amplified from RNA isolated from the prostate of a patient with benign prostatic hyperplasia using the primers hPSA-F1 5′-CCCAAGCTTACCACCTGCAC-3′ and hPSA-FLAG-Xho-R1 5′-TTTCTCGAGCTACTTGTCATCGTCGTCCTTGTAATCAGCGGGGTTGGCCACGATGGT-3′ and subcloned into the PCaG-Neo vector (Wako Pure Chemical Industries). The PSA–FLAG vector was then transiently transfected into CHO-K1 cells. After transfection, recombinant PSA was purified using a FLAG-tag system (Sigma, St. Louis, MO, USA) from serum free media. S2,3PSA and S2,6PSA containing asialo-type standard protein was purified utilizing an ACG lectin column and gel filtration column chromatography.

The expression plasmid for the α-DG373(T322R)-Fc was constructed as previously described (20). In this plasmid vector, the coding sequence of human α-DG (1-373) with amino acid substitution of threonine with arginine at position 322 was subcloned into pEF-Fc. Expression vector of Flag-tagged TagD was constructed as previously described (24). In the expression plasmid for Flag-tagged PCYT2 (Uniprot KB: Q99447-1) recombinant protein, the coding sequence of human PCYT2 with flag peptide (DYKDDDDK) purchased from addgene (#81074) was cloned into the pCAG-Neo vector (WAKO). The expression plasmids for Myc-tagged CDS1 (UniProt KB: P98191), CDS2 (UniProt KB: Q99L43), PCYT1A (UniProt KB: P49586), and PCYT1B (UniProt KB: Q811Q9) recombinant proteins were purchased from Origene. In bacterial expression plasmids for His6-tagged PCYT2β (Uniprot KB: Q99447-1) and PCYT2α (Uniprot KB: Q99447-3) recombinant proteins, the coding sequences were cloned into pColdI vector (Takara Bio Inc., Shiga, Japan).

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding wPDPN (accession No.: XM_007104824.2) plus an N-terminal RIEDL tag, which is recognized by an anti-RIEDL tag mAb (LpMab-7), was subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan). This plasmid was named pCAG-Neo/RIEDL-wPDPN. DNA encoding the CD20 gene (IRAL012D02) was provided by the RIKEN BRC through the National BioResource Project of MEXT, Japan. The open reading frame of CD20 plus the inserted RIEDL tag between Pro169 and Ala170 of human CD20 was subcloned into a pCAG-Ble vector using In-Fusion HD Cloning Kit. This plasmid was named pCAG-Ble/CD20-₁₆₉RIEDL₁₇₀.