Epitope Mapping of Anti-Bear Podoplanin Monoclonal Antibody PMab-247

Introduction

Podoplanin (PDPN)/T1alpha/Aggrus is a type I transmembrane sialoglycoprotein that is expressed in normal tissues, such as renal corpuscles, type I alveolar cells of lung, and lymphatic endothelial cells of every organ.⁽¹⁾ PDPN induces platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2).⁽²⁾ The interaction between PDPN on lymphatic endothelial cells and CLEC-2 on platelets facilitates the separation of embryonic blood/lymphatic vessels.⁽³⁾ Human PDPN is overexpressed in several malignant tumors, such as malignant mesotheliomas,^(4,5) oral squamous cell carcinomas,⁽⁶⁾ malignant brain tumors,⁽⁷⁾ and lung cancers.⁽⁸⁾ PDPN expression has also been associated with malignant progression and cancer metastasis.⁽⁹⁾

In our previous study, we used the Cell-Based Immunization and Screening (CBIS) method to develop sensitive and specific monoclonal antibodies (mAbs) against bear PDPN (bPDPN) to facilitate the immunohistochemical (IHC) analysis of paraffin-embedded tissue sections.⁽¹⁰⁾ Flow cytometry revealed that PMab-247 (mouse IgG₁, kappa), one of the established clones, recognized CHO/bPDPN but did not react with CHO-K1. Western blotting demonstrated that PMab-247 could detect bPDPN as a 48-kDa band in CHO/bPDPN cells. IHC analyses revealed that PMab-247 strongly stained pulmonary type I alveolar cells, lymphatic endothelial cells, and podocytes of the kidney.⁽¹¹⁾ This study aimed to determine the binding epitope of PMab-247 to bPDPN using enzyme-linked immunosorbent assay (ELISA) and IHC analyses.

Materials and Methods

Results and Discussion

We first synthesized a series of bPDPN peptides, which are summarized in Table 1. Using ELISA, PMab-247 detected the 68–87 peptide corresponding to the 68th–87th amino acids (AAs) of bPDPN. We then synthesized point mutants of 68–87 peptides (Table 2). Using ELISA, PMab-247 strongly detected P68A, I69A, N70A, R71A, E72A, G73A, V74A, T75A, L77A, L79A, D81A, T83A, T84A, E85A, S86A, and T87A, and weakly detected R82A. Conversely, it did not detect D76A, R78A, and E80A, indicating that Asp76, Arg78, Glu80, and Arg82 are the critical epitopes of PMab-247.

We then performed a blocking assay using IHC to confirm the PMab-247 epitope. PMab-247 reacted with lung type I alveolar cells (Fig. 1A, B). These reactions were completely neutralized by P68A (Fig. 1C, D). Conversely, R78A did not block these reactions of PMab-247 (Fig. 1E, F). Likewise, D76A, E80A, and R82A did not inhibit the PMab-247 reaction (Supplementary Fig. S1). Negative control (blocking buffer) did not show any staining (Fig. 1G, H). As shown in Figure 2A and B, PMab-247 reacted with the podocytes and Bowman's capsules of the kidneys. The reactions of PMab-247 in the kidneys were completely neutralized by P68A (Fig. 2C, D) although R78A did not block these reactions of PMab-247 (Fig. 2E, F). Negative control (blocking buffer) did not show any staining (Fig. 2G, H). These IHC results indicate that Asp76, Arg78, Glu80, and Arg82 of bPDPN are essential for PMab-247 binding to bPDPN.

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FIG. 1.

IHC analyses of bear lungs using PMab-247 and point mutants of bPDPN. Histological sections of the bear lungs were directly autoclaved in citrate buffer for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the sections were incubated with (A, B) PMab-247 (5 μg/mL), (C, D) PMab-247 (5 μg/mL) + P68A (50 μg/mL), (E, F) PMab-247 (5 μg/mL) + R78A (50 μg/mL), or (G, H) control (blocking buffer), followed by treatment with the Envision + Kit. (I, J) HE staining. Scale bar: 100 μm. bPDPN, bear podoplanin; HE, hematoxylin and eosin; IHCs, immunohistochemical.

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FIG. 2.

IHC analyses of bear kidneys using PMab-247 and point mutants of bPDPN. Histological sections of the bear kidneys were directly autoclaved in citrate buffer for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the sections were incubated with (A, B) PMab-247 (5 μg/mL), (C, D) PMab-247 (5 μg/mL) + P68A (50 μg/mL), (E, F) PMab-247 (5 μg/mL) + R78A (50 μg/mL), or (G, H) control (blocking buffer), followed by treatment with the Envision + Kit. (I, J) HE staining. Scale bar: 100 μm.

In summary, the critical epitopes of PMab-247 are Asp76, Arg78, Glu80, and Arg82 of bPDPN. Among these AAs, Glu80 and Arg82 are included in PLAG-like domains of bPDPN, which is important for PDPN/CLEC-2 interaction (Fig. 3). These findings can be applied to the production of more functional anti-bPDPN mAbs.

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FIG. 3.

Schematic illustration of the epitope recognized by PMab-247. PLAG and PLD. Red AAs indicate the critical epitope of PMab-247. AAs, amino acids; PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain.