Determination of critical epitope of PcMab-47 against human podocalyxin

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

We previously produced a novel anti-PODXL mAb, PcMab-47 (IgG 1 , kappa) [13]. PcMab-47 reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. We engineered PcMab-47 into 47-mG 2a , a mouse IgG 2a -type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG 2a -f, a core fucose-deficient type of 47-mG 2a , to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG 2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration than the former. In vitro analysis showed that 47-mG 2a -f exhibited a much stronger ADCC than 47-mG 2a against OSCC cells. In vivo analysis revealed that 47-mG 2a -f, but not 47-mG 2a , exerted an antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times. Although 47-mG 2a and 47-mG 2a -f exerted antitumor activities in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG 2a -f showed a higher antitumor activity than 47-mG 2a . These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibodybased therapy against PODXL-expressing OSCCs. Although engineered mAbs of PcMab-47 show high antitumor activities against cancer cells, the critical epitope of PcMab-47 remains to be identified.
In this study, we clarified the binding epitope of PcMab-47 using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemistry.

Plasmid preparation
The cDNA encoding the full-length open reading frame (ORF) of PODXL was obtained by PCR using cDNA derived from the LN229 cell line (ATCC) as a template. Appropriate oligonucleotides were used as primers to make each deletion mutants. PCR products were subcloned into pCAG vector (FUJIFILM Wako Pure Chemical Industries Ltd., Osaka, Japan) with signal sequence and PA tag using the In-Fusion PCR Cloning kit (Takara Bio, Inc., Shiga, Japan). All amino acid number were consistent with the NCBI Reference Sequence, NP_005388.2.

Results and discussion
We previously developed PcMab-47, a novel anti-PODXL mAb which exhibits a high specificity and sensitivity against human PODXL. PcMab-47 was shown to be useful for immunohistochemical analyses using paraffin-embedded tissues [13,14]. Furthermore, engineered mAbs of PcMab-47 exerted high antitumor activities against cancer cells in mouse xenograft models. Therefore, the determination of the binding epitope of PcMab-47 is critical to further develop a molecular targeting therapy against PODXL.
Finally, we performed a blocking assay using immunohistochemistry. PcMab-47 reacted with oral cancer tissues (Fig. 3A) and the reaction was completely neutralized by the wild-type peptide ( 200-TPTSSGHDHLMKISSSSSTV -219 ) and H206A. D207A did not block the reaction of PcMab-47, and this confirmed the results of the flow cytometric blocking assays.
Taken together, our results indicate that the critical epitope of PcMab-47 is Asp207, His208, Leu209, and Met210. Our findings could lead to the production of more functional anti-PODXL mAbs, which would be advantageous for antitumor effects against PODXL-expressing cancers.