MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 2, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0002

Short Communications

Anti-Podocalyxin Monoclonal Antibody 47-mG2a Detects
Lung Cancers by Immunohistochemistry

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

Lung cancer is one of the leading causes of cancer-related deaths in the world. Regardless of the advances inlung cancer treatments, the prognosis is still poor. Podocalyxin (PODXL) is a highly glycosylated type I
transmembrane protein that is expressed in normal tissues, including the heart, pancreas, and breast. It is also
found and used as a diagnostic marker in many cancers, such as renal, brain, breast, oral, and lung cancers.
We previously developed specific and sensitive anti-PODXL monoclonal antibodies, PcMab-47 (mouse IgG1,kappa) and its mouse IgG2a-type (47-mG2a), both of which were suitable for immunohistochemical analysesof oral cancers. In this study, we investigated the utility of PcMab-47 and 47-mG2a for the immunohistochemical analyses of lung cancers. PcMab-47 stained 51/70 (72.9%) cases of lung cancer, whereas 47-mG2astained 59/70 (84.3%) cases, indicating that the latter antibody is more sensitive and is useful for detecting
PODXL in lung cancers.
Keywords: podocalyxin, PODXL, monoclonal antibody, lung cancer

Podocalyxin (PODXL) is a highly N-glycan- and O-glycanglycosylated type I transmembrane protein with a molecularweight of 150,000–200,000.(9–12) It was originally discovered
in renal podocytes and is expressed in many kinds of tissues,
including the heart, pancreas, and breast. PODXL is also foundin cancer tissues.(13) It has been reported to be a diagnostic
marker and a prognostic indicator in some cancers, such as
renal,(14) brain,(12) breast,(15) oral,(16) and lung cancers.(17,18)
This protein is involved in tumor growth, invasion, and metastasis,(19) and its overexpression can cause undesirable outcomes. Furthermore, Kusumoto et al. reported that PODXL
overexpression induces epithelial–mesenchymal transition inlung AC and contributes to tumor progression.(20) Therefore,
more precise tools for detecting PODXL in lung cancer arenecessary for optimal analysis and treatment.
Previously, we established specific and sensitive antiPODXL mAbs, PcMab-47 (mouse IgG1, kappa), and mouse
IgG2a-type (47-mG2a).(21) Herein, we investigated the utilityof these two antibodies for immunohistochemical analysis oflung cancers.

Introduction

Lung cancer is one of the leading causes of cancerrelated deaths in the world.(1) The number of new casesand deaths respectively reaches about 1.8 and 1.6 million
per year. There are two main subtypes: small-cell lung cancer (SCLC; 15%) and non-SCLC (NSCLC). NSCLC includes
adenocarcinoma (AC), squamous cell carcinoma (SCC), andlarge cell carcinoma.(2) Regardless of the advances in lungcancer treatment, the prognosis is still poor with 5-year survival rates at approximately 15%.(3) In the early stages, lungcancer is treated via surgical removal, which can be complemented by radiotherapy and/or chemotherapy. In the advanced stages, many types of anticancer agents, including
cisplatin and docetaxel, are used for chemotherapy. In recentyears, some techniques for the detection and identificationof cell types and genetic mutations have been establishedthat enable accurate investigations. Some molecular targeteddrugs have displayed efficacy for lung cancer under specific conditions and have been approved for treatment. These
drugs include bevacizumab,(4) a mouse-human IgG1 chimericantibody against vascular endothelial growth factor; nivolumab,(5) a fully human IgG4 monoclonal antibody (mAb)against programmed cell death 1 (PD-1); pembrolizumab,(6,7)
a mouse-human IgG4 chimeric antibody against PD-1; andatezolizumab,(8) a mouse-human IgG1 chimeric antibody
against programmed cell death ligand-1.

Materials and Methods
Tissues

Cancer tissue microarrays of lung cancers were purchased from US Biomax (Rockville, MD) and Cybrdi(Frederick, MD).

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
*These authors contributed equally to this work.

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YAMADA ET AL.

Antibodies

Results and Discussion(21)

PcMab-47 was developed as described previously.
Appropriate VH and VL cDNAs of mouse PcMab-47 and CH
and CL of mouse IgG2a were subcloned into pCAG-Bleand pCAG-Neo vectors (FUJIFILM Wako Pure Chemical
Industries Ltd., Osaka, Japan), respectively, to generate 47mG2a. Antibody expression vectors were transfected into
ExpiCHO-S cells using the ExpiCHO Expression System(Thermo Fisher Scientific, Inc., Waltham, MA). 47-mG2a waspurified using Protein G Sepharose (GE Healthcare BioSciences, Pittsburgh, PA).

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Immunohistochemical 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
primary mAbs for 1 hour at room temperature and thentreated using an Envision+ kit (Agilent Technologies, Inc.)for 30 minutes. Color development was performed using 3,3diaminobenzidine tetrahydrochloride (Agilent Technologies,Inc.) for 2 minutes. Sections were then counterstained withhematoxylin (FUJIFILM Wako Pure Chemical IndustriesLtd.). Staining intensity was evaluated as -, 1+, 2+, or 3+.

To produce PcMab-47 (mouse IgG1, kappa), we previously
immunized mice with recombinant PODXL purified from theculture supernatant of the extracellular domain of PODXLexpressing LN229 cells.(21) We further engineered PcMab-47
into a mouse IgG2a-type mAb (47-mG2a). This antibody exhibited a higher binding affinity than PcMab-47 for PODXLexpressing oral SCC (OSCC) cells. Immunohistochemicalanalysis of oral cancer tissues using these two antibodies
revealed that 47-mG2a stained OSCC cells in a cytoplasmic
pattern at a much lower concentration. PcMab-47 and 47mG2a detected PODXL in 163/201 (81.1%) and 197/201(98.0%) OSCC samples, respectively.
In this study, we performed immunohistochemical analyses using PcMab-47 and 47-mG2a against several typesof lung cancers. Typical staining results of PcMab-47 and 47mG2a against SCC, adenosquamous cell carcinoma (ASCC),
small-cell carcinoma (SC), AC, bronchioloalveolar carcinoma (BC), and papillary carcinoma (PC) are shown in Figure 1 and Supplementary Figures S1–S5. Both PcMab-47 and47-mG2a stained cancer cells of all types of lung cancer in acytoplasmic staining pattern. Furthermore, both PcMab-47
and 47-mG2a stained endothelial cells in lung cancers (Fig. 2).
As shown in Table 1 and Supplementary Table S1,
PcMab-47 stained 51/70 cases (72.9%) of lung cancers.

FIG. 1. Immunohistochemical analysis by anti-PODXL antibodies against lung SCCs. Sections (Case-7) were incubatedwith 5 mg/mL of PcMab-47 (A, D) and 0.5 mg/mL of 47-mG2a (B, E) for 1 hour at room temperature, followed bytreatment with the Envision+ kit for 30 minutes. Color development was performed using 3,3-diaminobenzidine tetrahydrochloride for 2 minutes. Sections were then counterstained with hematoxylin. (C, F) Hematoxylin & eosin staining;
scale bar = 100 mm.

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PODXL IN LUNG CANCER

93FIG. 2. Anti-PODXL antibodies detect endothelial cells in lung SCCs. Sections (Case-25) were incubated with 5 mg/mL ofPcMab-47 (A, D) and 0.5 mg/mL of 47-mG2a (B, E) for 1 hour at room temperature, followed by treatment with the Envision+
kit for 30 minutes. Color development was performed using 3,3-diaminobenzidine tetrahydrochloride for 2 minutes. Sections
were then counterstained with hematoxylin. (C, F) Hematoxylin & eosin staining; scale bar = 100 mm; Arrows, endothelial cells.

Among them, 35/41 cases (85.4%) of SCC, 6/10 cases(60%) of ASCC, 3/7 cases (42.9%) of SC, 4/5 cases (80%)
of AC, 3/5 cases (60%) of BC, and 0/2 cases (0%) of PC
were stained. In contrast, 47-mG2a stained 59/70 cases(84.3%) of lung cancers. Among them, 38/41 cases (92.7%)
of SCC, 8/10 cases (80%) of ASCC, 4/7 cases (51.7%) ofSC, 4/5 cases (80%) of AC, 4/5 cases (80%) of BC, and 1/2cases (50%) of PC were stained.
In conclusion, our immunohistochemical results demonstrate that 47-mG2a is significantly more sensitive than theoriginal PcMab-47 for the detection of PODXL in LSCCs andcould be applied to other SCCs in the future.

Table 1. Summary of Immunostaining Using PcMab-47 and 47-mG2aPcMab-47 immunostaining

Tumor
type

No.
of cases

-1+2+

SCC
ASCC
SCACBCPC
Total

41
10
7
5
52
70

64
4
12
2
19

16
6
3
120
28

110
0
3
10
1547-mG2a immunostaining

3+No. ofpositive cases-1+2+3+

No. ofpositive cases

800000835/41 (85.4%)
6/10 (60%)
3/7 (42.9%)
4/5 (80%)
3/5 (60%)
0/2 (0%)51/70 (72.9%)32311111124202121114221020

150021018

38/41 (92.7%)
8/10 (80%)
4/7 (57.1%)
4/5 (80%)
4/5 (80%)
1/2 (50%)59/70 (84.3%)

SCC, squamous cell carcinoma; ASCC, adenosquamous cell carcinoma; SC, small-cell carcinoma; AC, adenocarcinoma; BC,bronchioloalveolar carcinoma; PC, papillary carcinoma.
The intensity of staining was evaluated as -, 1+, 2+, 3+.

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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 Statement

The authors have no conflicts 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
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: January 11, 2018
Accepted: February 11, 2018