MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 5, 2017
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0031

Establishment of CMab-43, a Sensitive and SpecificAnti-CD133 Monoclonal Antibody, for Immunohistochemistry
Shunsuke Itai,1,2,* Yuki Fujii,3,* Takuro Nakamura,1,* Yao-Wen Chang,1 Miyuki Yanaka,1 Noriko Saidoh,1
Saori Handa,1 Hiroyoshi Suzuki,4 Hiroyuki Harada,2 Shinji Yamada,1 Mika K. Kaneko,1 and Yukinari Kato1,3,5

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic
stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small
cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal
intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors andas a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) andcharacterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed thefull length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the firstscreening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/
CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot
and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot
analysis. One of 11 clones, CMab-43 (IgG2a, kappa), showed a sensitive and specific reaction against colon cancer
cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.
Keywords: CD133, monoclonal antibody, immunohistochemistry, colon cancer
Introduction

C

ancer stem cells (CSCs) share many of the properties
of non-neoplastic stem cells. They are also characterized by extensive proliferation, self-renewal, invasion, metastasis, and drug resistance.(1–3) Side-population cells andseveral protein markers specific to CSCs have been developed to isolate CSCs from cancer tissues and to investigate
the CSC properties in cancer tissues.(4) These CSC markers
include CD133 and CD44.(1,5–12)
CD133, also known as prominin-1, was first described as acell surface marker on hematopoietic stem cells.(13) It is afive-transmembrane glycoprotein composed of an N-terminal
extracellular tail, two small cytoplasmic loops, two large
extracellular loops containing several potential glycosylationsites, and a short C-terminal intracellular tail.(14) CD133 hasbeen used as a marker to identify CSCs derived from primarysolid tumors.(1) Its expression is also used as a prognostic
marker of gliomas.(15)

Herein, we produced sensitive and specific monoclonal
antibodies (mAbs) against CD133, which can be used forflow cytometry, Western blot, and immunohistochemical
analysis.
Materials and Methods
Cell lines

LN229, HCT-116, Chinese hamster ovary (CHO)-K1,
Caco-2, and P3X63Ag8U.1 (P3U1) cell lines were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). LN229/CD133 and CHO/CD133 were producedby transfecting pCAG/PA-CD133-RAP-MAP into LN229
and CHO-K1 cells using Neon transfection system (Thermo
Fisher Scientific, Inc., Waltham, MA) and Lipofectamine
LTX (Thermo Fisher Scientific, Inc.), respectively. A few
days after transfection, PA tag-positive cells(16) were sorted
using a cell sorter (SH800; Sony Corp., Tokyo, Japan). CHOK1, CHO/CD133, and P3U1 cell lines were cultured in RPMI

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
3
Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
4
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
5
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
*These authors contributed equally to this work.
2ª Shunsuke Itai et al. 2017; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.

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1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and
LN229, LN229/CD133, HCT-116, and Caco-2 cell lines
were cultured in Dulbecco’s modified Eagle’s medium(DMEM) (Nacalai Tesque, Inc.), supplemented with 10%
heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque,Inc.) at 37C in a humidified atmosphere containing 5% CO2
and 95% air.
Production of hybridoma

Female 4-week-old BALB/c mice were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all of the animal
experiments described herein.
Mice were immunized using intraperitoneal (i.p.) injections of LN229/CD133 cells together with Imject Alum
(Thermo Fisher Scientific, Inc.). After several additional
immunizations, a booster injection of LN229/CD133 cells
was intraperitoneally administered 2 days before harvesting
spleen cells. Spleen cells were then fused with P3U1 cells
using PEG1500 (Roche Diagnostics, Indianapolis, IN) or

ITAI ET AL.

GenomONE-CF (Ishihara Sangyo Kaisha, Ltd., Osaka, Japan). The resulting hybridomas were grown in RPMI medium
supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.).
Culture supernatants were screened using flow cytometry(first screening), Western blot (second screening), and immunohistochemical analyses (third screening). MAbs were
purified from supernatants of hybridomas cultured inHybridoma-SFM medium (Thermo Fisher Scientific, Inc.)
using Protein G Sepharose 4 Fast Flow (GE Healthcare
United Kingdom, Ltd., Buckinghamshire, England).
Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (EDTA) (Nacalai
Tesque, Inc.). After washing with 0.1% bovine serum albumin (BSA)/phosphate buffered saline (PBS), the cells weretreated with 1 mg/mL of anti-CD133 mAb (clone CMab-43)
for 30 min at 4C and then with Alexa Fluor 488-conjugatedantimouse IgG (1:1000; Cell Signaling Technology, Inc.,
Danvers, MA). Fluorescence data were collected using
EC800 or SA3800 Cell Analyzers (Sony Corp.).

FIG. 1. Characterization of CMab-43. (A, B) Flow cytometry with CMab-43; cells were treated with 1 mg/mL of CMab-43
followed by Alexa Fluor 488-conjugated antimouse IgG; black line, negative control. (C) Western blot using CMab-43; celllysates (10 mg) were electrophoresed and proteins were transferred onto polyvinylidene difluoride (PVDF) membranes.
After blocking, membranes were incubated with 1 mg/mL of CMab-43 or anti-b-actin (AC-15) and then incubated withperoxidase-conjugated antimouse IgG.

MABS AGAINST CD133
Western blot analysis

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.) and proteins were thenelectrophoresed on 5%–20% polyacrylamide gels (Wako Pure
Chemical Industries, Ltd., Osaka, Japan) and were transferred
onto polyvinylidene difluoride (PVDF) membranes (Merck
KGaA, Darmstadt, Germany). After blocking with 4% skim
milk (Nacalai Tesque, Inc.), membranes were incubated with
1 mg/mL of anti-CD133 mAb (clone CMab-43) and anti-bactin (clone AC-15; Sigma-Aldrich Corp., St. Louis, MO), andthen with peroxidase-conjugated antimouse IgG (1:1000 diluted; Agilent Technologies, Inc., Santa Clara, CA), and were
finally developed using ImmunoStar LD (Wako Pure Chemical Industries, Ltd.) using a Sayaca-Imager (DRC Co., Ltd.,Tokyo, Japan).
Determination of binding affinity using flow cytometry

LN229/CD133 and Caco-2 cells (2 · 105) were suspended
in 100 mL of serially diluted mAbs (6–100 mg/mL), and Alexa
Fluor 488-conjugated antimouse IgG (1:200; Cell Signaling
Technology, Inc.) was then added. Fluorescence data werecollected using a cell analyzer (EC800; Sony Corp.). The
dissociation constants (KD) were calculated by fitting thebinding isotherms using the built-in one-site binding models in
GraphPad PRISM 6 (GraphPad software, Inc., La Jolla, CA).

233

with the CHO-K1 parental cells, indicating that CMab-43 is
specific for CD133 (Fig. 1A). CMab-43 also recognized endogenous CD133 in Caco-2 colon cancer cells, but did notreact with HCT-116 colon cancer cells (Fig. 1B). On Western
blot analyses against LN229, LN229/CD133, CHO-K1, CHO/
CD133, and Caco-2 cells, CMab-43 detected a strong signal at*100 kDa in LN229/CD133 and CHO/CD133, and did amoderate signal in Caco-2 cells, indicating that CMab-43 isvery useful in Western blot analyses (Fig. 1C). The broad band
revealed by CMab-43 is likely due to various glycosylation
forms of CD133, known to be highly glycosylated.(17)We further determined the binding affinity of CMab-43 for
Caco-2 and LN229/CD133 cells using flow cytometry (Fig. 2).
The calculated KD values for CMab-43 against LN229/CD133
and Caco-2 cells are 4.4 · 10-9 M and 2.6 · 10-9 M, respectively,
indicating a high affinity for CD133-expressing cell lines.
We investigated the immunohistochemical utility ofCMab-43 in human colon cancers as high CD133 expression
was observed in Caco-2 colon cancer cell lines (Fig. 1C).
Recent studies have shown CD133 expression with luminal
staining.(18–20) Staining of intraglandular debris was also
observed in colorectal cancers. As shown in Figure 3 andSupplementary Figure 1, CMab-43 showed a luminal membrane expression pattern, and intraglandular debris was also

Immunohistochemical analyses

Colon cancer tissues were purchased from U.S. Biomax,
Inc. (Rockville, MD). Histological sections of 4-mm thickness were deparaffinized in xylene and were then rehydrated
and autoclaved in citrate buffer (pH 6.0; Agilent Technologies, Inc.) for 20 minutes. Sections were then incubated with
1 mg/mL of CMab-43 for 1 hour at room temperature andwere then treated using an Envision+ kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3,3diaminobenzidine tetrahydrochloride (Agilent Technologies,Inc.) for 2 minutes, and sections were then counterstainedwith hematoxylin (Wako Pure Chemical Industries, Ltd.).
Results

In this study, we immunized mice with LN229/CD133
cells followed by a booster i.p. injection of LN229/CD133
cells. Culture supernatants were screened using flow cytometry to assess reactions with LN229 and LN229/CD133
cells. A total of 100 clones were generated after limiting
dilution. Subsequently, mAbs were selected according totheir signal efficacy on Western blot analysis. These analyses
identified 79 of the 100 clones that were able to detect
CD133, of which 11 exerted a strong and specific reaction.
The 11 clones selected by Western blot analysis were further
tested, and immunohistochemical analysis demonstrated that
4 clones showed strong staining, 4 showed moderate staining,and 3 showed no staining against colon cancer tissues. Threeof the four clones that showed a strong staining against coloncancer tissues were determined to be of the IgG1 subclass; thefourth clone, CMab-43, was of the IgG2a subclass and wasselected for the subsequent study.
Flow cytometry demonstrated that CMab-43 reacts with
LN229/CD133 cells but not with LN229 brain tumor cells(Fig. 1A). CMab-43 reacted with CHO/CD133 cells but not

FIG. 2. Binding affinity of CMab-43 was determined using flow cytometry. LN229/CD133 and Caco-2 cells weresuspended in 100 mL of serially diluted CMab-43 (6 ng/mL–
100 mg/mL), and secondary antimouse IgG was then added.
Fluorescence data were collected using a cell analyzer.

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ITAI ET AL.

FIG. 3. Immunohistochemical analysis by CMab-43 against colon cancer tissues. (A, B) Sections were incubated with
1 mg/mL of CMab-43 for 1 hour at room temperature followed by treatment with Envision+ kit for 30 minutes. Color wasdeveloped using 3,3-diaminobenzidine tetrahydrochloride for 2 minutes, and sections were then counterstained with hematoxylin. (C, D) Hematoxylin & eosin staining; scale bar = 100 mm.
stained. Normal mucosae were not positive for CMab-43
staining (Supplementary Fig. S2). CMab-43 stained 5/6 cases(83.3%) of colon adenocarcinomas (Supplementary Fig. S2and Supplementary Table S1), indicating that CMab-43 isuseful for immunohistochemical analysis.
Discussion

Recently, we produced antipodoplanin (PDPN) cancerspecific mAbs clone LpMab-2(21,22) and LpMab-23,(23,24)
which specifically recognize cancer-type PDPN in tumor tissues. For this technology, it is critical that immunogens areproduced using cancer cell lines, such as LN229 glioblastoma
cells, which express cancer-specific glycan-attached membrane proteins. This method can be used to develop usefulmAbs against multiple membrane proteins. Using the same
method, we successfully developed clone CMab-43 of IgG2a
subclass, which is very useful for Western blot, flow cytometry, and immunohistochemical analysis against CD133.
Mouse IgG2a subclass possesses several advantages overmouse IgG1 subclass, including antibody-dependent cellularcytotoxicity and complement-dependent cytotoxicity, andIgG2a is also easy to purify using Protein-A or Protein-G.(25,26)
Using LN229/CD133 cells for immunization and firstscreening, purification of recombinant CD133 proteins is not
required. This method may be applicable for developing
mAbs, especially against membrane proteins that cannot beeasily purified. CMab-43 was highly efficacious in Western
blot analyses and produced strong staining in colon cancer
cells in immunohistochemical analysis. Hence, CMab-43
will likely be an advantageous tool for the pathological
identification of CD133 in many cancers.

Acknowledgments

We thank Yoshimi Nakamura for excellent technical assistance. This work was supported, in part, by the Basic
Science and Platform Technology Program for Innovative
Biological Medicine from Japan Agency for Medical Research and development, AMED (Y.K.). This work was also
supported, in part, by the Platform Project for Supporting
Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED (Y.K.), by project for utilizing
glycans in the development of innovative drug discovery
technologies from AMED (Y.K.), by the Platform for Drug
Discovery, Informatics, and Structural Life Science (PDIS)
from AMED (Y.K.), and by JSPS KAKENHI Grant Numbers
17K07299 (M.K.K.) and 16K10748 (Y.K.). This work was
performed, in part, under the Cooperative Research Programof Institute for Protein Research, Osaka University, CR-1705, and by the Grant for Joint Research Project of the Instituteof Medical Science, the University of Tokyo. The authors
thank Enago (www.enago.jp) for the English language review.
Author Disclosure Statement

Y. K. received research funding from Ono PharmaceuticalCo., Ltd. All other authors have nothing to disclose.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: June 2, 2017
Accepted: August 17, 2017