Epitope Mapping of PMab-225 an Anti-Alpaca Podoplanin Monoclonal Antibody Using Flow Cytometry

Introduction

Podoplanin (PDPN/T1alpha/Aggrus), a type I transmembrane glycoprotein, is expressed in normal tissues, such as lymphatic endothelial cells, type I lung alveolar cells, and renal podocytes.^(1–3) PDPN has been also known as a platelet aggregation-inducing factor in highly metastatic tumor cells.^(1,4) PDPN is frequently overexpressed in various malignant tumors, including several squamous cell carcinomas, mesothelioma, glioblastoma, and osteosarcoma.^(5–7) PDPN possesses three platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3; these domains exist at the N-terminus of PDPN.⁽⁸⁾ Furthermore, one or two PLAG-like domains (PLDs) have been reported to be present in the middle of PDPN.⁽⁹⁾ PLAG1–3 and/or PLD elicit tumor cell-induced platelet aggregation response through the activation of C-type lectin-like receptor 2 (CLEC-2) to facilitate cancer progression and metastasis.^(1,3) These results imply the importance of the developing anti-PDPN monoclonal antibodies (mAbs) for cancer therapeutic treatment.

Alpaca (lama pacos) is a livestock that is used in various applications in Latin America. This animal has also been used for the production of antigen-specific single domain antibodies (nanobodies).^(10,11) However, membrane proteins of alpaca have not been investigated due to the lack of specific antibodies. Because PDPN is important for blood/lymphatic vessel separation,^(12,13) high endothelial venule integrity,⁽¹⁴⁾ and embryonic cardiac development,⁽¹⁵⁾ sensitive and specific mAbs against alpaca PDPN (aPDPN) are necessary to analyze the expression and function of aPDPN. Recently, we developed a novel anti-aPDPN mAb, PMab-225, using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPN by flow cytometry, western blotting, and immunohistochemical analysis.⁽¹⁶⁾ However, the binding epitope of PMab-225 for aPDPN remains to be clarified. Thus, this study aimed to identify the epitope of PMab-225 using deletion mutants or point mutants of aPDPN.

Materials and Methods

Results

Ten deletion mutants and wild type (WT) of aPDPN were generated (Fig. 1A). Transient transfectants were produced using CHO-K1 cells, including WT (corresponding to 27–162 aa), dN30 (corresponding to 30–162 aa), dN40 (corresponding to 40–162 aa), dN50 (corresponding to 50–162 aa), dN60 (corresponding to 60–162 aa), dN70 (corresponding to 70–162 aa), dN80 (corresponding to 80–162 aa), dN85 (corresponding to 85–162 aa), dN90 (corresponding to 90–162 aa), dN95 (corresponding to 95–162 aa), and dN100 (corresponding to 100–162 aa). All deletion mutants and WT of aPDPN containing N-terminal RAP16 tag were analyzed using flow cytometry for the epitope mapping of PMab-225. PMab-2 (an anti-RAP16 tag mAb) detected all deletion mutants of aPDPN (Fig. 1B, left), indicating that the expression level of each deletion mutant is very high. In contrast, PMab-225 did not react with dN85, dN90, dN95, and dN100 (Fig. 1B, right), suggesting that N-terminus of PMab-225 epitope might exist between 80 aa and 85 aa of aPDPN.

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FIG. 1.

Epitope mapping of PMab-225 using deletion mutants of aPDPN. (A) Illustration of WT and 10 deletion mutants aPDPN: dN30, dN40, dN50, dN60, dN70, dN80, dN85, dN90, dN95, and dN100. Black bars: the positive reaction of PMab-225. White bars: the negative reaction of PMab-225. (B) Deletion mutants of aPDPN were analyzed using flow cytometry. Deletion mutants were incubated with PMab-2 (anti-RAP16 tag; left panel, red line), PMab-225 (anti-aPDPN mAb; right panel, blue line), or buffer control (black line) for 30 minutes at 4°C, followed by secondary antibodies. aPDPN, alpaca podoplanin; mAb, monoclonal antibody; PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; WT, wild type.

Next, to identify the binding epitope of PMab-225, we produced a series of point mutations of aPDPN in CHO-K1 cells, including E80A, D81A, L82A, P83A, T84A, A85G, E86A, S87A, T88A, V89A, H90A, and V91A. PMab-2 reacted with all point mutants (Fig. 2A). In contrast, PMab-225 reacted with all these mutants, except for T84A (Fig. 2B), indicating that Thr84 of aPDPN could be included in the critical epitope of PMab-225.

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FIG. 2.

Epitope mapping of PMab-225 using point mutants of aPDPN. (A, B) Transient point mutants expressing E80A, D81A, L82A, P83A, T84A, A85G, E86A, S87A, T88A, V89A, H90A, and V91A of aPDPN were treated with PMab-2 (A) (red line), PMab-225 (B) (blue line), or buffer control (A, B) (black line) for 30 minutes at 4°C, followed by secondary antibodies. (C) Schematic illustration of the epitope recognized by PMab-225. Underlined amino acid is a critical epitope of PMab-225.

Discussion

We have demonstrated that critical epitope of PMab-225 could be Thr84 of aPDPN using deletion mutants and point mutants of aPDPN. This result does not conclude that only Thr84 is a PMab-225-binding amino acid. In our experiences, the binding epitope of mAbs has been determined to be 5–13 aa^(18,19); therefore, PMab-225 might bind to the peptide or the glycopeptide around Thr84 of aPDPN. We should perform X-ray crystallography to exactly determine the binding epitope of PMab-225 to aPDPN in the future study.

We have developed mAbs against many mammalian PDPNs, such as human,⁽²⁰⁾ mouse,⁽²⁰⁾ rat,⁽²¹⁾ rabbit,⁽²²⁾ dog,⁽²³⁾ cat,⁽²⁴⁾ pig,^(25,26) bovine,⁽²⁷⁾ horse,⁽²⁸⁾ Tasmanian devil,⁽²⁹⁾ tiger,⁽³⁰⁾ and bear⁽³¹⁾ PDPNs. Almost all mAbs against PDPNs have been reported to react with the PLAG1–3 domains or PLD.^(9,24,32) Actually, Thr84 of aPDPN exists in PLD (Fig. 2C). In the future study, we should investigate whether PMab-225 could neutralize the aPDPN-induced platelet aggregation.

In conclusion, Thr84 of aPDPN, which is located in PLD, is critical for PMab-225-specific binding to aPDPN. PMab-225 could be a useful tool for elucidating the pathophysiological function of aPDPN.