Establishment of an Anticetacean Podoplanin Monoclonal Antibody PMab-237 for Immunohistochemical Analysis Yukinari Kato, 1,2, * Yoshikazu Furusawa, 1,2, * Shunsuke Itai, 1,3, * Junko Takei, 1,3 Takuro Nakamura, 1 Masato Sano, 1 Hiroyuki Harada, 3 Shinji Yamada, 1 and Mika K. Kaneko 1 Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological di- agnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG 1 , kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN. Keywords: whale podoplanin, PDPN, lymphatic endothelial cells, PMab-237 Introduction P odoplanin (PDPN), a type I transmembrane sialo- glycoprotein, is expressed in many cell types, including pulmonary type I alveolar cells, renal epithelial cells such as podocytes, and lymphatic endothelial cells of every organ. (1) PDPN has been used to distinguish lymphatic endothelial cells from vascular endothelial cells in pathophysiological studies. (2) As an endogenous receptor of PDPN, C-type lectin-like receptor-2 (CLEC-2) was previously reported in our studies. (3,4) The PDPN-CLEC-2 interaction facilitates the separation of embryonic blood and lymphatic vessels. (5) The expression of human PDPN (hPDPN) has been re- ported in many malignant tumors, including oral squamous cell carcinomas, (6) lung squamous cell carcinomas, (7) esophageal squamous cell carcinomas, (8) malignant brain tumors, (9) and malignant mesotheliomas. (10) The expres- sion of hPDPN is associated with malignant progression and cancer metastasis. (11) We recently developed monoclonal antibodies (mAbs) against human, (9) mouse, (12) rat, (13) rabbit, (14) bovine, (15) dog, (16) cat, (17) pig, (18) and horse (19) PDPNs. An anti-cat PDPN (cPDPN) mAb (PMab-52) was shown to cross-react with tiger PDPN (tigPDPN). (20) An anti-bovine PDPN (bovPDPN) mAb (PMab- 44) recognized not only bovPDPN but also goat, (21) sheep, (22) and alpaca (23) PDPNs. In this study, we immunized mice with CHO/whale PDPN (wPDPN) cells and established mAbs against wPDPN. Materials and Methods Cell lines P3X63Ag8U.1 (P3U1) and CHO-K1 cells were obtained from the American Type Culture Collection (ATCC, Mana- ssas, VA). Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding wPDPN (accession no.: XM_007104824.2) plus an N-terminal RIEDL tag, which are recognized by an anti-RIEDL tag mAb (LpMab-7), was subcloned into a pCAG- Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Plasmids were transfected using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific, Inc., Wal- tham, MA). Stable transfectants were selected by limiting di- lution and cultivation in a medium containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto, Japan). The P3U1, CHO-K1, CHO/wPDPN, CHO/hPDPN, (24) CHO/mouse PDPN (mPDPN), (24) CHO/rat PDPN (rPDPN), (13) CHO/rabbit PDPN (rabPDPN), (14) CHO/dog PDPN (dPDPN), (16) CHO/bovPDPN, (15) CHO/cPDPN, (17) CHO/- pig PDPN (pPDPN), (25) CHO/horse PDPN (horPDPN), (26) CHO/tigPDPN, (20) CHO/alpaca PDPN (aPDPN), (23) 1 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan. 2 New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan. 3 Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. *Equally contributed. MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 38, Number 3, 2019 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2019.0013 108 Downloaded by Yukinari Kato from www.liebertpub.com at 06/13/19. For personal use only.