Development of an anti-bear podoplanin monoclonal antibody PMab-247 for immunohistochemical analysis

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues because PDPN is known as a lymphatic endothelial cell maker in pathology. Recently, we established anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, horse, goat, tiger, alpaca, and Tasmanian devil. However, anti-bear PDPN (bPDPN) has not been established yet. In this study, we immunized mice with bPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/bPDPN) cells, and screened mAbs against bPDPN using flow cytometry. One of the mAbs, PMab-247 (IgG1, kappa), specifically detected CHO/bPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-247 for the functional analyses of bPDPN.


Introduction
A type I transmembrane sialo-glycoprotein, podoplanin (PDPN), is expressed in many cell types, such as renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells of every organ [1]. PDPN has been reported to distinguish lymphatic endothelial cells from vascular endothelial cells in pathophysiological studies [2]. C-type lectinlike receptor-2 (CLEC-2) was previously reported as an endogenous receptor of PDPN in our studies [3,4]. The PDPN-CLEC-2 interaction facilitates the separation of embryonic blood and lymphatic vessels [5]. Human PDPN (hPDPN) is expressed in many malignant tumors, including brain tumors [6] and mesotheliomas [7], and is associated with malignant progression and cancer metastasis [8].

Animals
Female BALB/c mice (6 weeks of age) were purchased from CLEA Japan (Tokyo, Japan). The animals were housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all animal experiments.

Flow cytometry
The cells were harvested following a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and treated with primary mAbs for 30 min at 4°C. Thereafter, the cells were treated with Alexa Fluor 488conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) or Oregon Green anti-rat IgG (1:2000; Thermo Fisher Scientific Inc.). Then, fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).

Results
Two mice were immunized with CHO/bPDPN cells (Fig. 1). The procedure in this study included four additional immunizations of CHO/bPDPN cells together with the Imject Alum (one mouse) or chitosan (one mouse) as adjuvants, followed by a final booster injection of CHO/bPDPN cells together with the Imject Alum (one mouse) or chitosan (one mouse). The developed hybridomas were seeded into 96-well plates and cultivated for 9 days. Wells positive for CHO/bPDPN and negative for CHO-K1 were selected by flow cytometry. The first screening approach identified strong signals from CHO/bPDPN cells and weak or no signals from CHO-K1 cells in 24 of the 480 wells (5.0%) for Imject Alum (one mouse) or in 4 of the 480 wells (0.83%) for chitosan (one mouse), indicating that Imject Alum was found to be a better adjuvant for this immunization. After several additional screenings, PMab-247 (IgG 1 , kappa) was finally selected from a mouse using chitosan.
PMab-247 recognized CHO/bPDPN cells, but showed no reaction with CHO-K1 cells, as assessed by flow cytometry (Fig. 2). PMab-247 did not cross-react with the other PDPNs (Fig. 3). The expression levels of PDPNs were confirmed by each positive control mAb.

Discussion
In this study, we established PMab-247 against bPDPN, which is suitable for flow cytometry, Western blot, and immunohistochemical analyses using CBIS method. Previously, we tried to develop anti-bPDPN mAbs using CBIS method, and added Imject Alum as an adjuvant only in a first injection in the same way with our previous studies [14,[24][25][26]. However, we could not establish anti-bPDPN mAbs, which are useful for immunohistochemical analyses (data not shown). Herein, we used Imject Alum or chitosan as adjuvants for every immunization, and successfully obtained high positive rates in flow cytometry, indicating that repeated use of adjuvants is also advantageous in CBIS method. The epitope of PMab-247 needs further investigation to clarify the sensitivity and specificity of PMab-247 against bPDPN.
We believe that PMab-247 should prove to be useful in elucidating the pathophysiological functions of bPDPN in some bear tumors such as osteosarcomas [27,28] or squamous cell carcinoma [29].

Conflicts of interest
Y.K. received research funding from ZENOAQ RESOURCE CO., LTD. The other authors have no conflict of interest.