MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 3, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0003

47-mG2a: A Mouse IgG2a-Typeof PcMab-47 Useful for Detecting Podocalyxin
in Esophageal Cancers
by Immunohistochemistry

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Mika K. Kaneko,* Shunsuke Itai,* Shinji Yamada, and Yukinari Kato

Esophageal cancer is one of the highly malignant cancers. It comprises two of the most common histologicaltumor types: squamous cell carcinoma (SCC) and adenocarcinoma. SCC accounts for about 90% of esophageal
cancers. Despite developments in treatment strategies, the prognosis and survival rate remain poor. Podocalyxin(PODXL) is a highly glycosylated type-I transmembrane protein. It is expressed in normal tissues such as
kidney, heart, breast, and pancreas. Upregulation of PODXL correlates with tumor progression, invasion, andmetastasis. Therefore, this glycoprotein could be a potential biomarker for predicting the prognosis of somecancers, for instance, brain, colorectal, oral, lung, bladder, prostate, and ovarian cancers. We previously developed a specific and sensitive anti-PODXL monoclonal antibody (mAb), PcMab-47 (mouse IgG1, kappa) andits mouse IgG2a-type (47-mG2a). We showed their utility in immunohistochemical analysis of oral cancers.
Herein, we demonstrate that PcMab-47 and 47-mG2a can also be used to detect esophageal squamous cell
carcinoma (ESCC) with this technique. These two antibodies, respectively, stained 123/130 (94.6%) and 127/130 (97.7%) ESCC cases, indicating that they can detect PODXL with high sensitivity in this carcinoma. Of
more than 3+ cases, 47-mG2a was more effective than PcMab-47, respectively, staining 56/127 (44.1%) and 41/123 (33.3%). Therefore, 47-mG2a can be used for the detection of PODXL in ESCC using immunohistochemical analysis.
Keywords: podocalyxin, PODXL, monoclonal antibody, esophageal cancer

IntroductionE

sophageal cancer is one of the highly malignant tumors with respect to prognosis and mortality rate. It isthe eighth most common cancer and most common cause ofcancer-related death. More than 450,000 new cases and400,000 deaths have been reported worldwide.(1,2) Esophageal cancer comprises two common histological types:
squamous cell carcinoma (SCC) and adenocarcinoma (AC).
SCC accounts for about 90% of esophageal cancers.(3,4)
Despite improvement in treatment strategies, prognosis remains poor with overall 5-year survival rates ranging from
15% to 25%.(5) This cancer is treated by surgery that can becomplemented by radiotherapy with or without chemotherapy. Preoperative neoadjuvant chemotherapy with cisplatin
accompanied by 5-fluorouracil can be effective and is regarded as standard treatment.(6,7) In contrast, adjuvant therapy is ineffective in many cases.(8,9) Several clinical trials

testing the treatment potential of monoclonal antibodies(mAbs) against esophageal cancer are ongoing.(10–12) However, few molecular targeting drugs against this disease havebeen approved.
Podocalyxin (PODXL) is a type-I transmembrane protein
highly glycosylated with N-glycan or O-glycan(12) belonging
to the CD34 family of sialomucins. It is expressed in normal
tissues, such as kidney, heart, breast, and pancreas. PODXL
upregulation has been correlated with tumor progression, invasion, and metastasis.(13) It is used as a diagnostic marker innumerous cancers, including brain,(14) colorectal,(15) oral,(16)lung,(17,18) bladder,(19) prostate,(20) and ovarian cancers.(21) Indeed, Borg et al. reported that PODXL expression in esophageal
AC is an independent prognostic biomarker for poor overall
survival.(22) Therefore, specific and sensitive anti-PODXL mAbs
are required.
We previously established anti-PODXL mAbs PcMab-47(mouse IgG1, kappa)(23) and mouse IgG2a-type (47-mG2a).(24) InDepartment of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
*Both these authors contributed equally to this work.

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PODXL IN ESOPHAGEAL CANCER

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FIG. 1. Immunohistochemical analysis by anti-PODXL antibodies against esophageal squamous cell carcinoma (Case26). Sections were incubated with 5 mg/mL of PcMab-47 (A, D) and 0.5 mg/mL 47-mG2a (B, E) for 1 hour at room
temperature followed by treatment with Envision+ kit for 30 minutes. Color was developed using 3,3-diaminobenzidine
tetrahydrochloride (DAB) for 2 minutes, and sections were then counterstained with hematoxylin. (C, F) HE staining; scalebar = 100 mm. HE, hematoxylin and eosin; PODXL, podocalyxin.
this study, we investigated their utility for immunohistochemical
analysis of esophageal squamous cell carcinoma (ESCC).

(Thermo Fisher Scientific, Inc., Waltham, MA). 47-mG2a
was purified using Protein G-Sepharose (GE Healthcare BioSciences, Pittsburgh, PA).

Materials and Methods
Tissues

Cancer tissue microarrays of esophageal cancers werepurchased from Cybrdi, Inc. (Frederick, MD; Cat. No. CC0201-001): Case-1–62; and, US Biomax, Inc. (Rockville, MD;
Cat. No. BC02011): Case-63–130.
Antibodies

PcMab-47, a mouse anti-PODXL mAb (IgG1, kappa), wasdeveloped as previously described.(23) Appropriate VH andVL complementary DNAs (cDNAs) of mouse PcMab-47 andCH and CL of mouse IgG2a were subcloned into pCAG-Bleand pCAG-Neo vectors (FUJIFILM Wako Pure ChemicalIndustries Ltd., Osaka, Japan), respectively, to generate 47mG2a.(24) Antibody expression vectors were transfected intoExpiCHO-S cells using the ExpiCHO Expression SystemImmunohistochemical analyses

Histologic sections (4 mm thick) were deparaffinized inxylene and then rehydrated and autoclaved in citrate buffer(pH 6.0; Agilent Technologies, Inc., Santa Clara, CA) for 20
minutes. Sections were then incubated with 0.5 or 5 mg/mL

Table 1. Summary of Immunostaining
Using PcMab-47 and 47-mG2aImmunostaining

PcMab-47
47-mG2aNo. ofcases-1+2+3+No. of
positivecases

1301307350313240

4156

123/130 (94.6%)
127/130 (97.7%)

The intensity of staining was evaluated as -, 1+, 2+, 3+.

160

primary mAbs for 1 hour at room temperature and treatedusing an Envision+ kit (Agilent Technologies, Inc.) for 30
minutes. Color was developed using 3,3-diaminobenzidine
tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and sections were counterstained with hematoxylin(FUJIFILM Wako Pure Chemical Industries Ltd.). The intensity of staining was evaluated as -, 1+, 2+, or 3+.

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Results and Discussion

Previously, we immunized mice with recombinant
PODXL and produced PcMab-47 (mouse IgG1, kappa).(23)This antibody was then engineered into a mouse IgG2a-type
mAb (47-mG2a).(24) 47-mG2a exhibited a much higher
PODXL-binding affinity compared with PcMab-47. Immunohistochemical analysis of oral cancer tissues revealed
that those mAbs sensitively stain cells in a cytoplasmic pattern. In this study, we performed immunohistochemical
analysis using PcMab-47 and 47-mG2a against ESCC.
Representative immunohistochemistry results of PcMab47 and 47-mG2a against ESCC are shown in Figure 1. Bothantibodies stained cancer cells in a cytoplasmic pattern(Fig. 1). As shown in Table 1 and Supplementary Table S1,PcMab-47 stained 123/130 (94.6%) and 47-mG2a 127/130(97.7%) of ESCCs, indicating that both antibodies can beused to detect PODXL in ESCC using this method. Amongmore than 3+ cases, 47-mG2a was more sensitive thanPcMab-47 with 56/127 (44.1%) and 41/123 (33.3%) occurrences, respectively (Table 1). In conclusion, 47-mG2a can beused to detect PODXL in ESCC by immunohistochemicalanalysis.
Acknowledgments

We thank Takuro Nakamura, Miyuki Yanaka, NorikoSaidoh, Saori Handa, and Yoshimi Nakamura for their excellent technical assistance. This research was supported by
AMED under Grant Numbers: JP17am0301010 (Y.K.),
JP17am0101078 (Y.K.), and JP17ae0101028 (Y.K.).
Author Disclosure StatementThe authors have no conflict of interest.
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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinari-k@bea.hi-ho.ne.jp
Received: January 13, 2018
Accepted: March 14, 2018