MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0048
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A Defucosylated Mouse Anti-CD10 Monoclonal Antibody(31-mG2a-f) Exerts Antitumor Activity in a Mouse
Xenograft Model of CD10-Overexpressed Tumors

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Hiroki Kawabata,1 Hiroyuki Suzuki,2 Tomokazu Ohishi,3 Manabu Kawada,3Mika K. Kaneko,1 and Yukinari Kato1,2

CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. It is expressed on
predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Therefore, antibodies against
CD10 are used for diagnosing follicular lymphoma and solid tumors, including renal carcinomas. In this study,
we developed an anti-human CD10 monoclonal antibody, clone C10Mab-31 (IgG1, kappa), which detects CD10
by flow cytometry and shows high affinity for CD10-overexpressed CHO-K1 (CHO/CD10) cells. Furthermore,
the defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) exhibits antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antitumor activities in mouse xenografts of CHO/CD10
cells. These results indicate that 31-mG2a-f exerts antitumor effects against CD10-expressing tumors and could
be valuable as part of an antibody treatment regimen for them.
Keywords: CD10, monoclonal antibody, ADCC, CDC, antitumor activity

squamous cell carcinoma cells.(10) In melanomas, CD10protein expression level was found to be more in metastaticmelanomas than primary tumors.(11) In normal thyroid tissue,
its expression is not detected, but is highly expressed in follicular variants of papillary thyroid cancer.(12,13)
Clear cell renal cell carcinomas account for 65%–75% of
all malignant renal tumors. The World Health Organization
2016 Classification of renal tumors has included renal cell
carcinoma with leiomyomatous stroma in a category of
emerging/provisional entities of renal cell carcinoma. Immunohistochemically, the tumor cells are typically positive
for CD10 with cytokeratin 7.(14) These results suggest theusefulness of CD10 as a diagnostic marker of solid tumors.
Furthermore, CD10 was also identified as membraneneutral endopeptidase-24.11, playing critical roles in thehydrolysis of biologically active peptides and their inactivation.(15) CD10 cleaves at the amino side of hydrophobic
residues of several peptide hormones, including glucagon,
enkephalins, substance P, neurotensin, oxytocin, and bradykinin.(16,17) In addition to its enzymatic activity, CD10Introduction

C

D10 is a type II transmembrane glycoprotein and has
been identified as a common acute lymphoblastic leukemia antigen.(1–3) It is expressed on predifferentiated lymphocyte progenitor cells, and is detected on activated andproliferating B cells in the germinal center(4) and on bonemarrow stromal cells and neutrophils.(5) CD10 is also expressed in human B-cell malignancies, including follicular,(6)Burkitt, and lymphoblastic lymphomas.(7) Therefore, antibodies against CD10 are used for the diagnosis of follicularlymphoma.(6) Moreover, CD10 is expressed in normal epithelial tissues, including the small intestine, kidney, and lung.
A growing number of evidence suggests that elevatedCD10 expression is associated with the malignant progression of several solid tumors.(8) For example, CD10 overexpression has been associated with colorectal cancer
development and progression.(9) Furthermore, a direct correlation between CD10 expression and cell migration/anchorage-independent growth was observed in esophageal

Departments of 1Antibody Drug Development and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai, Japan.
3
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.

59

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60

regulates intracellular signaling pathways like PI3-kinase and
Fak-Src.(18) Moreover, the association between CD10 andthe tumor suppressor PTEN leads to decreased PIP3 phosphorylation, which activates the Akt pathway.(19) However,the detailed molecular functions of CD10 to promote tumor
progression have not been fully investigated.
CD10 could be a useful diagnostic marker and an attractive
molecular target for cancer therapy. The expression analysis
along with other markers might be helpful in tumor diagnosis
and treatment. Therefore, sensitive and specific monoclonal
antibodies (mAbs) against CD10 are needed to diagnose andtreat malignancies.
In this study, we developed a novel anti-human CD10
mAb, C10Mab-31 (IgG1, kappa), by immunizing mice with
the CD10-overexpressed human renal cancer OUR-10 cells.
C10Mab-31 can be used in flow cytometry. We further developed the defucosylated mouse IgG2a version of C10Mab31 (31-mG2a-f) and investigated the antitumor activities
against CD10-overexpressed tumors.
Materials and Methods

KAWABATA ET AL.
Hybridoma production

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 the animal
experiments described in this study. BALB/c mice were
immunized using intraperitoneal (i.p.) injections of OUR-10/
CD10 (1 · 108 cells) together with Imject Alum (Thermo
Fisher Scientific, Inc.). After several additional immunizations, a booster injection was intraperitoneally administered 2
days before harvesting spleen cells. Spleen cells were thenfused with P3U1 cells using PEG1500 (Roche Diagnostics,
Indianapolis, IN). The resulting hybridomas were grown in
RPMI 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement(Thermo Fisher Scientific, Inc.). Culture supernatants werescreened using flow cytometry (CHO-K1 vs. CHO/CD10).
MAbs were purified from the supernatants of hybridomas,
cultured in Hybridoma-SFM medium (Thermo Fisher Scientific, Inc.) using Protein G Sepharose (GE Healthcare
Biosciences, Pittsburgh, PA, USA).

Cell lines

P3X63Ag8U.1 (P3U1) and CHO-K1 cells were obtained
from the American Type Culture Collection (ATCC; Manassas, VA). OUR-10 (a renal cancer cell line) was provided
from Cell Resource Center for Biomedical Research Instituteof Development, Aging and Cancer, Tohoku University(Miyagi, Japan). The pCAG-Ble vector and the pCAG-Neo
vector were purchased from FUJIFILM Wako Pure Chemical
Corporation (Osaka, Japan). OUR-10/CD10 and CHO/CD10
cells were established by transfecting pCAG-Ble/CD10PAtag and pCAG-Neo/CD10-PAtag plasmids (accession no.
NM_000902.3) into OUR-10 cells and CHO-K1 cells, respectively. P3U1, OUR-10, OUR-10/CD10, CHO-K1, andCHO/CD10 were cultured in RPMI 1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan), supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mL of penicillin, 100 lg/mLof streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai
Tesque, Inc.) at 37C in a humidified atmosphere containing
5% CO2 and 95% air.

Antibodies

To generate 31-mG2a, we subcloned VH cDNA of C10Mab31 and CH of mouse IgG2a into the pCAG-Neo vector, alongwith cDNA of C10Mab-31 light chain into the pCAG-Zeo
vector, respectively. The vector of 31-mG2a was transfected
into BINDS-09 cells (FUT8-knocked out ExpiCHO-S cells)
using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.).(20) The resulting mAb, 31-mG2a-f, was purified
with Protein G Sepharose (GE Healthcare Biosciences,
Pittsburgh, PA).

Animals

All animal experiments were performed following relevant
guidelines and regulations to minimize animal suffering anddistress in the laboratory. Animal experiments for antitumor
activity were approved by the Institutional Committee forExperiments of the Institute of Microbial Chemistry (permit
no. 2021-019 for antibody-dependent cellular cytotoxicity[ADCC] and permit no. 2021-009 for antitumor activities).
Mice were maintained in a specific pathogen-free environment (23C – 2C, 55% – 5% humidity) on an 11-hour light/
13-hour dark cycle with food and water supplied ad libitum
across the experimental period. Mice were monitored for
health and weight every 2–5 days during the 3-week period ofeach experiment. We determined the loss of original bodyweight to a point >25% and/or a maximum tumor size
>3000 mm3 as humane endpoints for euthanasia. Mice wereeuthanized by cervical dislocation; death was verified byrespiratory and cardiac arrest.

FIG. 1. Production of 31-mG2a (mouse IgG2a) and 31mG2a-f (defucosylated form) from an anti-CD10 mAb,
C10Mab-31 (mouse IgG1).

ANTITUMOR ACTIVITY OF ANTI-CD10 MAB
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/phosphate-buffered saline (PBS), the cells were treatedwith 1 lg/mL of anti-CD10 mAbs (C10Mab-31 and 31-mG2a-f)
for 30 minutes at 4C and subsequently with Alexa Fluor
488-conjugated anti-mouse IgG (1:1000; Cell Signaling
Technology, Inc., Danvers, MA). Fluorescence data werecollected using EC800 Cell Analyzers (Sony Corp., Tokyo,
Japan).
ADCC

mesh. Erythrocytes were effectively lysed by a 10-second
exposure to ice-cold distilled water. Splenocytes were washed with Dulbecco’s modified Eagle’s medium (DMEM)
and resuspended in DMEM with 10% FBS as effector cells.
Target cells were labeled with 10 lg/mL calcein AM (Thermo
Fisher Scientific, Inc.) and resuspended in the medium. The
target cells (2 · 104 cells/well) were placed in 96-well plates
and mixed with effector cells and either 31-mG2a-f or control
IgG (mouse IgG2a) (Sigma-Aldrich Corp., St. Louis, MO).
After a 5-hour incubation period, the calcein release of
supernatant from each well was measured. The fluorescenceintensity was determined at an excitation wavelength of
485 nm and an emission wavelength of 538 nm using a microplate reader (Power Scan HT) (BioTek Instruments, Winooski, VT). Cytolytic activity (as % of lysis) was calculatedusing the following formula: % lysis = (E - S)/(M - S) · 100(where E is the fluorescence released in the experimental
cultures of target and effector cells, S is the spontaneous

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Six 6-week-old female BALB/c nude mice were purchasedfrom Charles River, spleens were removed aseptically, andsingle-cell suspensions were obtained by dispersing the
spleens using a syringe and pressing through stainless steel

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FIG. 2. Flow cytometry using C10Mab-31 and 31-mG2a-f. (A) CHO-K1 and CHO/CD10 cells were treated with C10Mab31 and 31-mG2a-f, or buffer control, followed by secondary antibodies. (B) Determination of the binding affinity of
C10Mab-31 and 31-mG2a-f for CHO/CD10 cells using flow cytometry. CHO/CD10 cells were suspended in 100 mL ofserially diluted C10Mab-31 and 31-mG2a-f, followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG.
Fluorescence data were collected using the EC800 Cell Analyzer.

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fluorescence released in cultures containing only target cells,and M is the maximum fluorescence obtained by adding alysis buffer containing 0.5% Triton X-100, 10 mM Tris-HCl(pH 7.4), and 10 mM of EDTA to the target cells to lyse
all cells).
Complement-dependent cytotoxicity (CDC)

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CHO/CD10 cells were placed in 96-well plates of 2 · 104
cells/well in DMEM supplemented with 10% FBS. Cells
were incubated with either 31-mG2a-f or the control IgG(mouse IgG2a) (Sigma-Aldrich Corp.) and 10% of rabbit
complement (Low-Tox-M Rabbit Complement) (Cedarlane
Laboratories, Hornby, ON, Canada) for 5 hours at 37C. To
assess cell viability, MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
inner salt] assay was performed using a CellTiter 96 AQueous
assay kit (Promega, Madison, WI).
Antitumor activity of 31-mG2a-f

KAWABATA ET AL.

We first characterized anti-CD10 mAbs in flow cytometry.
Both C10Mab-31 and 31-mG2a-f reacted with CHO/CD10
cells, not with CHO-K1 cells, indicating that C10Mab-31 and
31-mG2a-f are specific for CD10 (Fig. 2A).
Kinetic analysis of C10Mab-31 and 31-mG2a-f interactions
with CHO/CD10 cells was conducted by flow cytometry. As
shown in Figure 2B, the KD for C10Mab-31 and 31-mG2a-f
interactions with CHO/CD10 cells was 5.7 · 10-9 M
and 2.1 · 10-9 M, respectively, suggesting that both C10Mab31 and 31-mG2a-f showed a high affinity for CHO/CD10
cells.
31-mG2a-f-mediated ADCC and CDC
in CHO/CD10 cells

We investigated whether 31-mG2a-f was capable of mediating ADCC against CHO/CD10 cells. As shown in
Figure 3, 31-mG2a-f showed ADCC (21.4% cytotoxicity)
against CHO/CD10 cells more effectively than the control
mouse IgG2a (9.9% cytotoxicity; p < 0.05).

Female BALB/c nude mice (6-week old) were purchasedfrom Charles River (Kanagawa, Japan) and used in experiments when they were 6 weeks old. CHO/CD10 (0.3 mL of
1.33 · 108/mL in DMEM) was mixed with 0.5 mL of BD
Matrigel Matrix Growth Factor Reduced (BD Biosciences,
San Jose, CA). A 100 lL suspension (containing 5 · 106
cells) was injected subcutaneously into the left flanks of nude
mice. After day 6, 100 lg of 31-mG2a-f and control mouse
IgG (Sigma-Aldrich Corp.) in 100 lL PBS were injected into
the peritoneal cavity of each mouse. Additional antibodies
were then injected on days 14 and 21. The tumor diameterand volume were determined as previously described.(21) The
mice were euthanized 25 days after cell implantation. Alldata were expressed as mean – standard error of the mean
(SEM).
Statistical analyses

All data are expressed as mean – SEM. Statistical analysis
was conducted with Welch’s t test for ADCC, CDC, andtumor weight. Analysis of variance (ANOVA) and Sidak’s
multiple comparisons tests were conducted for tumor volume
and mouse weight. All calculations were performed using
GraphPad Prism 8. A p-value of <0.05 was considered statistically significant.
Results
Flow cytometry analysis against CHO/CD10
cells using anti-CD10 mAbs

In this study, we immunized one mouse with OUR-10/
CD10 cells. Flow cytometry was conducted to check reactions with CHO-K1 and CD10-overexpressed CHO-K1(CHO/CD10) cells. A stronger reaction against CHO/CD10
was needed compared with CHO-K1. We obtained one clone,
C10Mab-31, of the IgG1 subclass. We converted the subclassof C10Mab-31 into mouse IgG2a to add ADCC and CDC
activities. Furthermore, we produced a defucosylated antiCD10 mAb (31-mG2a-f) using BINDS-09 cells (FUT8deficient ExpiCHO-S cells). This process is summarized in
Figure 1.

FIG. 3. Evaluation of ADCC and CDC elicited by 31mG2a-f. (A) ADCC elicited by 31-mG2a-f, control mouse
IgG2a targeting CHO/CD10 cells. (B) CDC elicited by
31-mG2a-f, control mouse IgG2a targeting CHO/CD10 cells.
Values are mean – SEM. Asterisks indicate statistical significance (*p < 0.05; Welch’s t-test). ADCC, antibody-dependent
cellular cytotoxicity; CDC, complement-dependent cytotoxicity; SEM, standard error of the mean.

ANTITUMOR ACTIVITY OF ANTI-CD10 MAB
31-mG2a-f-mediated antitumor activities against
the CHO/CD10 xenograft model

To study the antitumor activity of 31-mG2a-f on cell
growth in vivo, CHO/CD10 cells were subcutaneously implanted into the flanks of nude mice. 31-mG2a-f and control
mouse IgG were injected three times (on days 6, 14, and 21

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We then investigated whether 31-mG2a-f could mediate
CDC against CHO/CD10 cells. As shown in Figure 3, 31mG2a-f elicited a higher degree of CDC (51.3% cytotoxicity)
in CHO/CD10 cells compared with that elicited by control
mouse IgG2a (17.1% cytotoxicity; p < 0.05). These results
demonstrated that 31-mG2a-f promoted significantly higher
levels of ADCC and CDC against CHO/CD10 cells.

63

FIG. 4. Evaluation of antitumor activity of 31-mG2a-f in CHO/CD10 xenografts. (A) CHO/CD10 cells (5 · 106 cells) were
injected subcutaneously into the left flank. After day 6, 100 mg of 31-mG2a-f or control mouse IgG in 100 ml PBS were injected
intraperitoneally into mice; additional antibodies were then injected on days 14 and 21. The tumor volume was measured ondays 6, 11, 14, 18, 21, and 25 after the injection. Value are means – SEM. Asterisks indicate statistical significance (*p < 0.05,**p < 0.01; ANOVA and Sidak’s multiple comparisons test). (B) Tumors of CHO/CD10 xenografts were resected from 31mG2a-f and control mouse IgG groups. Tumor weight on day 25 was measured from excised xenografts. Values aremean – SEM. Asterisk indicates statistical significance (**p < 0.01, Welch’s t-test). (C) Resected tumors of CHO/CD10 xenografts from the control mouse IgG and 31-mG2a-f groups on day 25. Scale bar, 1 cm. (D) Body weights and appearance ofthe mice implanted with CHO/CD10 xenografts. Body weights of mice implanted with CHO/CD10 xenografts were recordedon days 6, 11, 14, 18, 21, and 25. (n.s., ANOVA and Sidak’s multiple comparisons test). (E) Body appearance of mice on day
25. Scale bar, 1 cm. ANOVA, analysis of variance; n.s., not significant; PBS, phosphate-buffered saline.

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64

after cell injections) into the peritoneal cavity of mice. Tumor
formation was observed in mice from the control and 31mG2a-f-treated groups in CHO/CD10 xenograft models. 31mG2a-f significantly reduced the tumor development of
CHO/CD10 xenograft compared with that in control mouse
IgG on days 14 ( p < 0.05), 18 ( p < 0.01), 21 ( p < 0.01), and25 ( p < 0.01) (Fig. 4A). The administration of 31-mG2a-f
resulted in a 90% reduction of tumor volume compared withthat of the control mouse IgG on day 25 postinjection.
The tumor weight of mice treated with 31-mG2a-f wassignificantly lower than that in the control mouse IgG groupin CHO/CD10 xenograft models (82% reduction; p < 0.01,
Fig. 4B). The resected tumors of CHO/CD10 xenografts are
depicted in Figure 4C. Body weight was not significantly
different among the two groups in CHO/CD10 xenograftmodels (Fig. 4D). CHO/CD10 xenograft mice models on day
25 are shown in Figure 4E.
Taken together, these results indicate that the administration of 31-mG2a-f effectively reduced the growth of
CHO/CD10 xenografts.
Discussion

MAbs to target solid tumor antigens have been extensively
explored.(22) Among the U.S. Food and Drug Administrationapproved mAbs, most mAbs receiving approval in solid tumors have targeted two members of the ERBB family, EGFR
or HER2.(23) In addition, the cytotoxic agent conjugatedmAbs against HER2,(24–26) Nectin-4,(27) and TROP2,(28,29)
have been approved by FDA in solid tumors. NonconjugatedmAbs can exhibit several different mechanisms of action,
including ADCC and CDC activities, while conjugated mAbs
rely on the direct cytotoxicity of their payloads through endocytosis of receptor-bound mAbs-drug conjugates.
This study showed that anti-CD10 mAb, 31-mG2a-f, exhibits ADCC and CDC activities and potent antitumor activity in CHO/CD10 xenografts. Also, a 90% reduction intumor volume and 82% reduction in tumor weight wereachieved (Fig. 4). These therapeutic outcomes depend on theisotype and specific nature of the mAb (i.e., specific binding
epitope, affinity of target binding, and particular conformation), as well as the nature of the target antigen. We have notdetermined the binding epitope; however, both C10Mab-31
and 31-mG2a-f showed high affinity to target (Fig. 2B), andthe defucosylation in 31-mG2a-f confers high ADCC activity.
These results expect the use of anti-CD10 mAb not only forpathological diagnosis but also for tumor therapy.
On the other hand, an important consideration in tumortargeted antibody-drug is the distribution of the target protein expression in normal tissues, which has important
implications for off-target effects. Previously, we establishedcancer-specific monoclonal antibodies (CasMabs) technology against cell surface proteins and developed some CasMabs, including anti-podoplanin mAb (LpMab-2)(30) andanti-podocalyxin (PcMab-60).(31) For example, LpMab-2 canrecognize both a Thr55-Leu64 peptide of human podoplaninand cancer-type aberrant glycosylation of Thr55 and/or
Ser56, unexpressed in normal cells.(30) Therefore, the establishment of CasMabs to CD10 is essential to reduce theside effects.
CD10 belongs to the mammalian neutral endopeptidases
and has a C-terminal extracellular domain that contains the

KAWABATA ET AL.

active site.(5,16,17) The X-ray crystal structure of the extracellular domain (52–749 aa) of CD10 was solved with ametalloproteinase inhibitor phosphoramidon. The structure
reveals the important consensus sequences HExxH and
ExxxD that embrace a large central cavity, including theactive site. The inhibitor is bound to this cavity, which
provides detailed ligand recognition.(32) However, there isno information about the epitope of C10Mab-31. Therefore,
further studies are warranted for determining the epitope. We
have developed two novel epitope mapping methods, such as
REMAP and HisMAP method, and determined the epitope of anti-EGFR mAbs (EMab-51 and EMab-134),(33,34) ananti-CD44 mAb (C44Mab-46),(35) and an anti-CD20 mAb(C20Mab-60).(36) Furthermore, based on the epitope analysis, the investigations of C10Mab-31 effects on the CD10mediated ligand recognition and endopeptidase activity arethought to be required.
Recently, the functional anti-CD10 mAb ( JMAM-1) has
been reported.(37) JMAM-1 has a cytostatic effect on mesothelioma MSTO-211H cells in vitro and exhibits a prolonged
survival of NCI-H226 tumor-bearing mice. However, thedetailed mechanism of the antitumor effect has not been investigated. Moreover, CD10-positive subpopulation in headand neck squamous cell carcinoma possesses cancer stem
cell-like properties and expresses a high level of stem cellmarker OCT3/4. The elevated level of OCT3/4 in CD10positive cells results in the promotion of stemness and tumor
formation, which implies therapeutic resistance and refractory.(38) Therefore, the functional investigations of C10Mab31 (or 31-mG2a-f) in endogenous CD10-expressing tumor
cells are required to understand the antitumor activity of
31-mG2a-f.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency
for Medical Research and Development (AMED) under
grant nos. JP22ama121008 (to Y.K.), JP21am0401013 (toY.K.), and JP21am0101078 (to Y.K.). and by the Japan Society for the Promotion of Science ( JSPS) Grants-in-Aid for
Scientific Research (KAKENHI) under grant nos. 21K07168(to M.K.K.) and 19K07705 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: September 27, 2021
Accepted: February 14, 2022