MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 2, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0002

Anti-CD133 Monoclonal Antibody CMab-43 Exerts
Antitumor Activity in a Mouse Xenograft
Model of Colon Cancer

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Yukinari Kato,1,2 Tomokazu Ohishi,3 Shinji Yamada,1 Shunsuke Itai,1 Yoshikazu Furusawa,1,2Masato Sano,1 Takuro Nakamura,1 Manabu Kawada,3 and Mika K. Kaneko1Cancer stem cells contribute to tumorigenesis, metastasis, recurrence, and chemoresistance. CD133/prominin1—a pentaspan membrane glycoprotein—has been used as a stem cell biomarker for the isolation of stemlike cells from a variety of normal and pathological tissues. In our previous studies, we developed several
anti-CD133 monoclonal antibodies using Cell-Based Immunization and Screening (CBIS) methods, followed by characterization of their efficacy by flow cytometry, western blotting, and immunohistochemicalanalyses. One of the 100 clones, CMab-43 (IgG2a, kappa), demonstrated a sensitive and specific reactionagainst colon cancer cells. This study aimed to investigate the antitumor activity of CMab-43. Caco-2 cells(human colon cancer cell line) were subcutaneously implanted into the flanks of nude mice. CMab-43 andcontrol mouse IgG were injected three times into the peritoneal cavity of mice. Tumor formation wasobserved in the control and CMab-43-treated mice of Caco-2 xenograft models. CMab-43 significantly
reduced tumor development of Caco-2 xenograft in comparison with the control mouse IgG on days 12, 14,and 17. Our results cumulatively suggest that CMab-43 is useful for antibody therapy against CD133expressing colon cancers.
Keywords: CD133, monoclonal antibody, colon cancers, Caco-2

In our previous studies, we developed novel anti-CD133
monoclonal antibodies (mAbs)(16) using Cell-Based Immunization and Screening (CBIS) method.(17–20) We firstexpressed the full length of CD133 in LN229 glioblastoma
cells, immunized mice with LN229/CD133 cells, and thenperformed the first screening by flow cytometry. After limiting
dilution, we established 100 anti-CD133 mAbs, reacting withLN229/CD133 cells but not with LN229 cells. Subsequently,
we performed the second and third screening, with western
blotting and immunohistochemical analyses, respectively.
Among the 100 mAbs, 11 strongly reacted with CD133 inwestern blotting analysis. One of the 11 clones, CMab-43(IgG2a, kappa), demonstrated a sensitive and specific reaction
against colon cancer cells. Among the mouse IgG subclasses,
IgG2a(21) and IgG2b(22) are known to possess antibody-dependent
cellular cytotoxicity (ADCC) and complement-dependent
cytotoxicity (CDC). In this study, we investigated theIntroduction

Cancer stem cells (CSCs) or tumor-initiating cells
possess several properties of non-neoplastic stem cells.
CSCs are also characterized by extensive proliferation, selfrenewal, invasion, metastasis, and chemoresistance.(1–3)
Several protein markers, such as CD133 and CD44 as well as
side-population cells, have been utilized to isolate CSCs fromcancerous tissues as well as to investigate the CSC properties
in cancer tissues.(1,4–12) CD133, also known as prominin-1,
was first described as a cell surface marker on hematopoietic
stem cells.(13) It is a pentaspan membrane glycoprotein,composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing
several potential glycosylation sites, and a short C-terminal
intracellular tail.(14) Its expression also serves as a prognostic
marker of gliomas.(15)12
3

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.

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antitumor activity of CMab-43 against mouse xenograftmodels of human colon cancer.
Materials and Methods
Cell lines

Caco-2 was obtained from the American Type CultureCollection (ATCC, Manassas, VA) and was cultured in theDulbecco’s modified Eagle’s medium (DMEM) medium(Nacalai Tesque, Inc., Kyoto, Japan) supplemented with
10% heat-inactivated fetal bovine serum (FBS;Thermo Fisher
Scientific, Inc., Waltham, MA), 100 U/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.

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Antibodies

The mouse anti-CD133 mAb CMab-43 was developed as
described elsewhere.(16) CMab-43 hybridoma was cultured inHybridoma-SFM medium (Thermo Fisher Scientific, Inc.),
supplemented with 100 U/mL of penicillin, 100 mg/mL ofstreptomycin, and 25 mg/mL of amphotericin B (NacalaiTesque, Inc.) at 37C under a humidified 5% CO2 and 95%air atmosphere. CMab-43 was purified using Protein GSepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA).
Control mouse IgG was purchased from Sigma-Aldrich Corp.
(St. Louis, MO).
Antitumor activity of CMab-43

Female BALB/c nude mice (6-week old) were purchasedfrom Charles River (Kanagawa, Japan) and used in experiments when they were 10 weeks old. Caco-2 (0.3 mL of1.33 · 108/mL in DMEM) were mixed with 0.5 mL of BD
Matrigel Matrix Growth Factor Reduced (BD Biosciences,San Jose, CA). A 100-mL suspension (containing 5 · 106cells) was injected subcutaneously into the left flanks of nude
mice. After day 1, 50 mg of CMab-43 and control mouse IgG
in 100 mL PBS were injected into the peritoneal cavity of eachmouse. Additional antibodies were then injected on days 7and 14. The tumor diameter and volume were determined aspreviously described.(23) The mice were euthanized 19 days
after cell implantation. All data were expressed as mean –SEM. Statistical analysis was performed using the Tukey–
Kramer’s test. p < 0.05 was considered to be statisticallysignificant.
Results and Discussion

We had previously reported the development of themouse mAb CMab-43 against CD133.(16) Flow cytometry
demonstrated that CMab-43 reacted with LN229/CD133
cells but not with LN229 brain tumor cells, which indicatesthat CMab-43 is specific for CD133. CMab-43 also recognized as an endogenous CD133 in Caco-2 colon cancercells. Western blotting against LN229, LN229/CD133,
CHO-K1, CHO/CD133, and Caco-2 cells revealed thatCMab-43 detected a strong signal at *100 kDa in LN229/FIG. 1. Antitumor activity of CMab-43 against Caco-2
xenograft model. Tumor volume of Caco-2 xenografts.
Caco-2 cells were injected subcutaneously into female nudemice. The indicated antibodies (50 mg/day; 2.5 mg/kg) wereadministered intraperitoneally on days 1, 7, and 14 after cell
inoculation. The tumor volume was measured at the indicated time points. The values are presented as mean – SEM.
*p < 0.01, Tukey–Kramer’s test; SEM, standard error of themean.

CD133 and CHO/CD133 but detected a moderate signal in
Caco-2 cells, which indicates the usefulness of CMab-43 inwestern blotting. Furthermore, the calculated KD values for
CMab-43 against LN229/CD133 and Caco-2 cells were
4.4 · 10-9 and 2.6 · 10-9 M, respectively, indicating a highaffinity for CD133-expressing cell lines. The immunohistochemical analysis demonstrated that CMab-43 stained
83.3% of colon adenocarcinomas, which indicated the usefulness of CMab-43 for immunohistochemical analysis.(16)As CMab-43 was determined to be an IgG2a subclass ofmouse IgG, it demonstrates the potential of ADCC and CDC
activities.(21)To study the antitumor activity of CMab-43 on cell growthin vivo, Caco-2 cells were subcutaneously implanted into theflanks of nude mice. CMab-43 and control mouse IgG wereinjected three times (on days 1, 7, and 14 after cell injections)
into the peritoneal cavity of mice. Tumor formation wasobserved in mice from the control and CMab-43-treatedgroups in Caco-2 xenograft models. CMab-43 significantly
reduced the tumor development of Caco-2 xenograft incomparison with that in control mouse IgG on days 12, 14,and 19 (Fig. 1). Caco-2 xenograft mice models on day 19 areshown in Figure 2A. The resected tumors of Caco-2 xenografts are depicted in Figure 2B. The tumor weight of mice inCMab-43 treated was significantly lower than that in thecontrol mouse IgG group in Caco-2 xenograft models(Fig. 2C). However, body weight was not significantly different among the two groups in the Caco-2 xenograft models(Fig. 2D).
In conclusion, CMab-43 is applicable for antibody therapy against human colon cancers expressing CD133.

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ANTI-CD133 MAB AGAINST COLON CANCER

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FIG. 2. Evaluation of antitumor activity of CMab-43 against Caco-2 xenograft model. (A) Caco-2 xenograft mice models
on day 19. (B) Resected tumors of Caco-2 xenografts. (C) Tumor weight of Caco-2 xenografts (day 19). (D) Body weight of
Caco-2 xenografts (day 19). The values are presented as mean – SEM. An asterisk indicates statistical significance.
*p < 0.01, Tukey–Kramer’s test; n.s., not significant.
Further studies on antitumor activities against CD133expressing xenografts are, therefore, necessary to obtain a moredetailed understanding of antibody therapy against CD133.
Acknowledgments

We thank Akiko Harakawa, Shun-ichi Ohba, Miyuki
Yanaka, Kayo Hisamatsu, Saori Handa, and Yoshimi Nakamura for their excellent technical assistance. This research was supported in part by AMED under grant nos.
JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), andJP18ae0101028 (Y.K.), and by JSPS KAKENHI grant no.
17K07299 (M.K.K.) and grant no. 16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp;
yukinari-k@bea.hi-ho.ne.jp
Received: January 18, 2019
Accepted: March 17, 2019