Development and characterization of anti‐glycopeptide monoclonal antibodies against human podoplanin, using glycan‐deficient cell lines generated by CRISPR/Cas9 and TALEN

Abstract Human podoplanin (hPDPN), which binds to C‐type lectin‐like receptor‐2 (CLEC‐2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer‐associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung‐type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti‐hPDPN mAb, LpMab‐21. To characterize the hPDPN epitope recognized by the LpMab‐21, we established glycan‐deficient CHO‐S and HEK‐293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab‐21 is Thr76–Arg79. LpMab‐21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab‐21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell‐type‐specific. LpMab‐21 combined with other anti‐hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN.

Furthermore, to characterize the hPDPN epitope recognized by the LpMab-21, we need glycan-deficient CHO-S or HEK-293T cell lines. We report the establishment of glycan-deficient cell lines using the CRISPR/Cas9 or TALEN.

Materials and Methods
Cell lines, mice, and human tissues As described in detail previously [36,39], the cell lines LN229, HEK-293T, NCI-H226, U-2 OS, Met-5A, Chinese hamster ovary (CHO)-K1, and P3U1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The HSC-2 and HSC-4 cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). The MG-63 cell line was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University (Miyagi, Japan). The LN319 cell line was provided by Prof. Kazuhiko Mishima (Saitama Medical University, Saitama, Japan) [40]. Human lymphatic endothelial cells (LECs), CHO-S cells, and PC-10 cells were purchased from Cambrex Corp., East Rutherford, NJ, Thermo Fisher Scientific Inc., (Waltham, MA), and Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan), respectively. LN229 and CHO-K1 cells were transfected with the hPDPN plasmids using Lipofectamine 2000 (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions [30].
Three female BALB/c mice (4-week-old) were purchased from CLEA Japan (Tokyo, Japan) and were housed under pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein.
The Tokyo Medical and Dental University Institutional Review Board and the Sendai Medical Center Review Board reviewed and approved the use of human cancer tissues. Written informed consent was obtained for using the human cancer tissue samples. Microarrays of normal human tissues were purchased from Cybrdi, Inc. (Frederick, MD).

Generation of deletion mutants
Amplified hPDPN cDNA was subcloned into a pCAG-Ble(Zeo) vector (Wako Pure Chemical Industries Ltd.) with a MAP-tag, detected by PMab-1 [41,42], which was added to the N-terminus using the In-Fusion HD Cloning Kit (Clontech, Palo Alto, CA). Deletion mutants of hPDPN were generated using the primers as follows: Sense primers and designation of the corresponding mutant Antisense primer 5′-TCTAGAGTCGCGGCCGCTTACTTGTCGTCATCGT CHO-K1 cells were transfected with these plasmids using Lipofectamine LTX (Thermo Fisher Scientific Inc.). Deletion mutants were cultured in RPMI 1640 medium containing l-glutamine (Nacalai Tesque, Inc.) and 10% heat-inactivated FBS at 37°C in a humidified atmosphere containing 5% CO 2 . Stable transfectants of CHO-K1/ssMAP-hPDPNdN mutants were selected by culturing them in medium containing 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).

Production of point mutants
The amplified hPDPN cDNA was subcloned into a pcDNA3 vector (Thermo Fisher Scientific Inc.), and a FLAG epitope tag was added to the C-terminus. Substitutions of amino acid residues to Ala or Gly in the hPDPN sequence were performed, using a QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies Inc., Santa Clara, CA) using oligonucleotides containing the desired mutations. CHO-S or CHO-K1 cells were transfected with the plasmids using a Gene Pulser Xcell electroporation system (Bio-Rad Laboratories Inc.). Point mutants were cultured in RPMI 1640 medium containing l-glutamine.

Flow cytometry
Cell lines were harvested after brief exposure to 0.25% Trypsin/1 mmol/L EDTA (Nacalai Tesque, Inc.). After washing with 0.1% BSA in PBS, the cells were treated with primary mAbs for 30 min at 4°C, followed by treatment with Oregon Green 488-conjugated to goat antimouse IgG or anti-rat IgG (Thermo Fisher Scientific Inc.). Fluorescence data were acquired using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Previously, we developed the original technology to produce cancer-specific mAbs that detect cell type-specific posttranslational modifications of the same protein [30]. We used LN229/hPDPN cells as the immunogen to elicit novel anti-PDPN mAbs. We produced several clones including LpMab-2, LpMab-3, and LpMab-9 as anti-glycopeptide mAbs [30]. Recently, we further immunized mice with LN229/hPDPN cells to develop further anti-glycopeptide mAbs against human PDPN, and characterized several clones, including LpMab-12 and LpMab-19. In this study, we characterized another clone LpMab-21, which detects many human cancer cell lines that express PDPN, such as those derived from glioblastomas, lung squamous cell carcinomas, oral squamous cell carcinomas, osteosarcomas, and malignant mesotheliomas. The isotypes of previously established anti-PDPN mAbs are IgG 1 (seven clones) and IgG 3 (one clone). However, the applications of mouse IgG 3 mAbs are limited because they often aggregate [52]. Moreover, mouse IgG 1 and IgG 3 isotypes do not induce ADCC or CDC. Therefore, we required chimeric mAbs using human IgG 1 to investigate these activities [33]. LpMab-21 (IgG 2a subclass) could be used to investigate the function of anti-tumor activities in xenograft models because LpMab-21 induced ADCC and CDC (data not shown).
Furthermore, we need several glycan-deficient cell lines such as sialic acid-deficient or N-glycan deficient cell lines to characterize those mAbs. In this study, we successfully produced several glycan-deficient cell lines such as sialic acid deficient (PDIS-14 and PDIS-22) or N-glycan deficient cell lines (PDIS-1, PDIS-9, and PDIS-12), using CRISPR/ Cas9 and TALEN systems. Using those cell lines, we determined that the epitope of LpMab-21 includes sialic acids, indicating that we can also investigate whether the epitope of novel mAbs against the other membrane proteins possesses sialic acids or N-glycans.
We showed here that LpMab-21 detected glioblastomas, oral cancers, and seminomas (Fig. 5) as well as normal cells such as lymphatic endothelial cells, basal epithelial cells of the esophagus, and myoepithelial cells of breast glands (Fig. 3). In contrast, LpMab-21 did not react with the renal glomerulus or with type I alveolar cells of lung (Fig. 3), indicating that sialylation of hPDPN is tissue-specific.
In conclusion, LpMab-21 shows promise for investigating the expression and function of hPDPN in cancers and normal tissues. Further, mAbs that recognize different epitopes of hPDPN should serve as powerful tools for identifying the function of hPDPN.